Genetically Engineered Swine Influenza Virus and Uses Thereof

(19) TZZ _T

(11) EP 2 497 492 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 39/00 (2006.01) A61K 39/12 (2006.01) 08.08.2018 Bulletin 2018/32 A61K 39/145 (2006.01) C12N 15/64 (2006.01) C12N 5/00 (2006.01) A61K 39/116 (2006.01) (21) Application number: 12163377.0

(22) Date of filing: 01.06.2005

(54) Genetically engineered swine influenza virus and uses thereof Genetisch veränderter Schweineinfluenzavirus und Verwendungen damit Virus de la grippe porcine génétiquement modifié et ses utilisations

(84) Designated Contracting States: • Webster, Robert AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Memphis, TN 38120 (US) HU IE IS IT LI LT LU MC NL PL PT RO SE SI SK TR • Lager, Kelly, M. Designated Extension States: Colo, IA 50056 (US) AL BA HR LV MK YU (74) Representative: Jones Day (30) Priority: 01.06.2004 US 576418 P Rechtsanwälte,Attorneys-at-Law, Patentanwälte Prinzregentenstrasse 11 (43) Date of publication of application: 80538 München (DE) 12.09.2012 Bulletin 2012/37 (56) References cited: (60) Divisional application: WO-A-99/64068 17209515.0 / 3 332 803 • WANG X ET AL: "Functional Replacement of the (62) Document number(s) of the earlier application(s) in Carboxy-Terminal Two-Thirds of the Influenza A accordance with Art. 76 EPC: Virus NS1 Protein with Short Heterologous 05856778.5 / 1 773 384 Dimerization Domains", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR (73) Proprietors: MICROBIOLOGY, US, vol. 76, no. 24, 1 December • Icahn School of Medicine at Mount Sinai 2002 (2002-12-01), pages 12951-12962, New York, NY 10029 (US) XP003006649, ISSN: 0022-538X • The United States of America as represented by • TALON J ET AL: "Influenza A and B Viruses the Expressing Altered NS1 Proteins: A Vaccine Secretary of Agriculture Approach", PROCEEDINGS OF THE NATIONAL Washington DC 20250-0302 (US) ACADEMY OF SCIENCES OF USA, NATIONAL • St. Jude Children’s Research Hospital ACADEMY OF SCIENCE, WASHINGTON, DC, US, Memphis, TN 38105 (US) vol. 97, no. 8, 1 April 2000 (2000-04-01), pages 4309-4314, XP002985464, ISSN: 0027-8424 (72) Inventors: • LANDOLT G A ET AL: "COMPARISON OF THE • Palese, Peter PATHOGENESIS OF TWO GENETICALLY New York, NY 10128 (US) DIFFERENT H3N2 INFLUENZA A VIRUSES IN • Garcia-Sastre, Adolfo PIGS",JOURNAL OF CLINICAL MICROBIOLOGY, New York, NY 10128 (US) WASHINGTON, DC, US, vol. 41, no. 5, 1 May 2003 • Webby, Richard, J (2003-05-01), pages 1936-1941, XP008075053, Memphis, TN 38112 (US) ISSN: 0095-1137 • Richt, Juergen, A. Manhattan, KS 66502 (US)

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 497 492 B1

Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 497 492 B1

• DATABASE EMBASE [Online] ELSEVIER • WANG X ET AL: "INFLUENZA A VIRUS NS1 SCIENCE PUBLISHERS, AMSTERDAM, NL; 1951, PROTEIN PREVENTS ACTIVATION OF SHOPE R E: "The provocation of masked swine NF-KAPPAB AND INDUCTION OF ALPHA/BETA influenza virus by infection with human influenza INTERFERON", JOURNAL OF VIROLOGY, THE virus", XP002559476, Database accession no. AMERICAN SOCIETY FOR MICROBIOLOGY, US, EMB-0007087537 & TIJDSCHRIFT VOOR vol. 74, no. 24, 1 December 2000 (2000-12-01), DIERGENEESKUNDE 1951, vol. 76, no. 11, 1951, pages 11566-11573, XP001181389, ISSN: pages 414-420, ISSN: 0040-7453 0022-538X, DOI: • WEBBY RICHARD J ET AL: "Evolution of swine 10.1128/JVI.74.24.11566-11573.2000 H3N2 influenza viruses in the United States", • RICHT JÜRGEN A ET AL: "Vaccination of pigs September 2000 (2000-09), JOURNAL OF against swine influenza viruses by using an VIROLOGY, VOL. 74, NR. 18, PAGE(S) 8243-8251, NS1-truncated modified live-virus vaccine.", XP002559477, ISSN: 0022-538X * page 8244; table JOURNAL OF VIROLOGY NOV 2006, vol. 80, no. 1 * 22, November 2006 (2006-11), pages • DATABASE MEDLINE [Online] US NATIONAL 11009-11018, ISSN: 0022-538X LIBRARY OF MEDICINE (NLM), BETHESDA, MD, • KHIABANIAN HOSSEIN ET AL: "Reassortment US;June 2005 (2005-06), SOLÓRZANO ALICIA ET patterns in Swine influenza viruses.", PLOS ONE AL: "Mutations in the NS1 protein of swine 07 OCT 2009, vol. 4, no. 10, 7 October 2009 influenza virus impair anti-interferon activity and (2009-10-07), page e7366, ISSN: 1932-6203 confer attenuation in pigs.", XP002559478, Database accession no. NLM15919908 & JOURNAL OF VIROLOGY JUN 2005, vol. 79, no. 12, June 2005 (2005-06), pages 7535-7543, ISSN: 0022-538X • DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; November 2007 (2007-11), VINCENT AMY L ET AL: "Efficacy of intranasal administration of a truncated NS1 modified live influenza virus vaccine in swine", XP002559479, Database accession no. PREV200800070215 & VACCINE, vol. 25, no. 47, November 2007 (2007-11), pages 7999-8009, ISSN: 0264-410X

2 EP 2 497 492 B1

Description

1. FIELD OF THE INVENTION

5 [0001] The present invention relates, in general, to a genetically engineered attenuated swine influenza viruses haying an impaired ability to antagonize the cellular interferon (IFN) response, and the use of such attenuated virus in vaccine and pharmaceutical formulations. In particular, the invention relates to a genetically engineered attenuated swine influenza viruses having modifications to a swine influenza virus A/Swine/Texas/4199-2/98 NS1 gene that diminish or elim- inate the ability of the NS1 gene product to antagonize the cellular IFN response. These viruses replicatein vivo, but 10 demonstrate decreased virulence and increased attenuation, and therefore are well suited for use in live virus vaccines, and pharmaceutical formulations.

2. BACKGROUND

15 2.1 Influenza Virus

[0002] Virus families containing enveloped single-stranded RNA of the negative-sense genome are classified into groups having non-segmented genomes (Paramyxoviridae, Rhabdoviridae, Filoviridae and Borna Disease Virus) or those having segmented genomes (Orthomyxoviridae, Bunyaviridae and Arenaviridae). The Orthomyxoviridae family, 20 described in detail below, and used in the examples herein, includes the viruses of influenza, types A, B and C viruses, as well as Thogoto and Dhori viruses and infectious salmon anemia virus. [0003] The influenza virions consist of an internal ribonucleoprotein core (a helical nucleocapsid) containing the single- stranded RNA genome, and an outer lipoprotein envelope lined inside by a matrix protein (M1). The segmented genome of influenza A virus consists of eight molecules (seven for influenza C) of linear, negative polarity, single-stranded RNAs 25 which encode eleven polypeptides, including: the RNA-dependent RNA polymerase proteins (PB2, PB1 and PA) and nucleoprotein (NP) which form the nucleocapsid; the matrix membrane proteins (M.1, M2); two surface glycoproteins which project from the lipid containing envelope: hemagglutinin (HA) and neuraminidase (NA); the nonstructural protein (NS 1), nuclear export protein (NEP); and the proapoptotic factor PB1-F2. Transcription and replication of the genome takes place in the nucleus and assembly occurs via budding on the plasma membrane. The viruses can reassort genes 30 during mixed infections. [0004] Influenza virus adsorbs via HA to sialyloligosaccharides in cell membrane glycoproteins and glycolipids. Fol- lowing endocytosis of the virion, a conformational change in the HA molecule occurs within the cellular endosome which facilitates membrane fusion, thus triggering uncoating. The nucleocapsid migrates to the nucleus where viral mRNA is transcribed. Viral mRNA is transcribed by a unique mechanism in which viral endonuclease cleaves the capped 5’- 35 terminus from cellular heterologous mRNAs which then serve as primers for transcription of viral RNA templates by the viral transcriptase. Transcripts terminate at sites 15 to 22 bases from the ends of their templates, where oligo(U) sequences act as signals for the addition of poly(A) tracts. Of the eight viral RNA molecules so produced, six are mono- cistronic messages that are translated directly into the proteins representing HA, NA, NP and the viral polymerase proteins, PB2, PB1 and PA. The other two transcripts undergo splicing, each yielding two mRNAs which are translated 40 in different reading frames to produce M1, M2, NS1 and NEP. The PB1 segment encodes a second protein, the nonstructural PB1-F2 protein, by using of an alternative ATG. In other words, the eight viral RNA segments code for eleven proteins: nine structural and two nonstructural. A summary of the genes of the influenza virus and their protein products is shown in Table I below.

45 TABLE I : INFLUENZA VIRUS GENOME RNA SEGMENTS AND CODING ASSIGNMENTSa Length Length Encoded d Molecules Segment b (Amino Comments (Nucleotides) Polypeptide Per Virion c Acids) RNA transcriptase component; host cell 50 1 2341 PB2 759 30-60 RNA cap binding RNAtranscriptase component; initiation 2 2341 PB1 757 30-60 of transcription PB1-F2 87 Proapoptotic factor 55 3 2233 PA 716 30-60 RNA transcriptase component

3 EP 2 497 492 B1

(continued)

Length Length Encoded d Molecules Segment b (Amino Comments (Nucleotides) Polypeptidec Per Virion 5 Acids) Hemagglutinin; trimer; envelope 4 1778 HA 566 500 glycoprotein; mediates attachment to cells Nucleoprotein; associated with RNA; 10 5 1565 NP 498 1000 structural component of RNA transcriptase Neuraminidase; tetramer; envelope 6 1413 NA 454 100 glycoprotein

15 7 1027 M1 252 3000 Matrix protein; lines inside of envelope Structural protein in plasma membrane; M 96 ? 2 spliced mRNA

8 890 NS1 230 Nonstructural protein;function unknown NEP 121 ? Nuclear export protein; spliced mRNA 20 a Adapted from R.A. Lamb and P.W. Choppin (1983), Annual Review of Biochemistry, Volume 52, 467-506. b For A/PR/8/34 strain c Determined by biochemical and genetic approaches d Determined by nucleotide sequence analysis and protein sequencing

25 [0005] The pathogenicity of influenza viruses is modulated by multiple virus and host factors. Among the host factors that fight virus infections, the type I interferon (IFN α/β) system represents a powerful antiviral innate defense mechanism which was established relatively early in the evolution of eukaryotic organisms (García-Sastre, 2002, Microbes Infect 4:647-55). The antiviral IFNα/β system involves three major steps: (i) detection of viral infection and IFNα/β secretion, 30 (ii) binding of IFNα/β to its receptors and transcriptional induction of IFNα/β-stimulated genes, and (iii) synthesis of antiviral enzymes and proteins. Most viruses, however, have acquired specific genetic information encoding IFNα/β antagonist molecules, which effectively block one or more steps of the antiviral IFNα/β system. Influenza A viruses express a non-structural protein in infected cells, the NS1 protein (described in detail,infra ), which counteracts the cellular IFNα/β response (García-Sastre et al., 1998, Virology 252:324-30). 35 [0006] The influenza A virus genome contains eight segments of single-stranded RNA of negative polarity, coding for two nonstructural and nine structural proteins. The nonstructural protein NS 1 is abundant in influenza virus infected cells, but has not been detected in virions. NS1 is a phosphoprotein found in the nucleus early during infection and also in the cytoplasm at later times of the viral cycle (King et al., 1975, Virology 64: 378). Studies with temperature-sensitive (ts) influenza mutants carrying lesions in the NS gene suggested that the NS1 protein is a transcriptional and post- 40 transcriptional regulator of mechanisms by which the virus is able to inhibit host cell gene expression and to stimulate viral protein synthesis. Like many other proteins that regulate post-transcriptional processes, the NS1 protein interacts with specific RNA sequences and structures. The NS1 protein has been reported to bind to different RNA species including: vRNA, poly-A, U6 snRNA, 5’ untranslated region as of viral mRNAs and ds RNA (Qiu et al., 1995, RNA 1: 304; Qiu et al., 1994, J. Virol. 68: 2425; Hatada Fukuda 1992, J Gen Virol. 73:3325-9. Expression of the NS1 protein 45 from cDNA in transfected cells has been associated with several effects: inhibition of nucleo-cytoplasmic transport of mRNA, inhibition of pre-mRNA splicing, inhibition of host mRNA polyadenylation and stimulation of translation of viral mRNA (Fortes, et al., 1994, EMBO J. 13: 704; Enami et al., 1994, J. Virol. 68: 1432; de la Luna et al., 1995, J. Virol. 69:2427; Lu et al., 1994, Genes Dev. 8:1817; Park et al., 1995, J. Biol Chem. 270,28433; Nemeroff et al., 1998, Mol. Cell. 1:1991; Chen et al., 1994, EMBO J. 18:2273-83). In particular, the NS1 protein has three domains that have been 50 reported to have a number of regulatory functions during influenza virus infection. The amino-terminal 73 amino acids are responsible for binding to RNAs (Qian et al., 1995, RNA 1:948-956), particularly double stranded RNAs, conferring to the virus the ability to escape the interferon α/β response (Donelan et al., 2003, J. Virol. 77:13257-66). The central portion of the protein interacts with the eukaryotic translation initiation factor 4GI facilitating preferential translation of viral mRNAs over host mRNAs (Aragón et al., 2000, Mol. Cell Biol, 20:6259-6268). The carboxy-terminus or the effector 55 domain has been shown to inhibit host mRNA processing, specifically, inhibition of host mRNA polyadenylation (Nemeroff et al., 1998, Mol. Cell 1:991-1000), binding to poly(A) tails of mRNA inhibiting nuclear export (Qiu and Krug, 1994, J. Virol. 68:2425-2432) and inhibition of pre-mRNA splicing (Lu et al., 1994, Genes Dev. 8:1817-1828). [0007] Studies of human recombinant influenza virus lacking the NS1 gene (deINS1) showed that this virus could only

4 EP 2 497 492 B1

replicate in IFN-incompetent systems such as STAT1-/- mice or Vero cells; thus the NS1 protein is responsible for IFN antagonist activity (Garcia-Sastre et al., 1998, Virology 252:324-330). Also, it has been shown that human influenza viruses with truncated NS1 proteins are attenuated in mice (Egorov et al., 1998, J. Virol. 72:6437-6441) and provide protection against wild-type challenge (Talon et al., 2000, Proc. Natl. Acad. Sci. USA 97:4309-4314). 5 2.2 Swine Influenza Virus

[0008] Swine influenza (SI) is an acute respiratory disease of swine caused by type A influenza viruses. Its severity depends on many factors, including host age, virus strain, and secondary infections (Easterday, 1980, Philos Trans R 10 Soc Lond B Biol Sci 288:433-7). Influenza A viruses are segmented negative-strand RNA viruses and can be isolated from a number of otheranimal host species, including birds, humans, horses, whales, and mink. Although whole influenza viruses rarely cross the species barrier, gene segments can cross this barrier through the process of genetic reassortment, or genetic shift. Since pigs support the replication of both human and avian influenza A viruses (Kida et al., 1994, J Gen Virol 75:2183-8), they have been postulated to play an important role in interspecies transmission by acting as a "mixing 15 vessel" for reassortment between viruses specific to different host species (Scholtissek, 1994, Eur J Epidemiol 10:455-8). This may lead to the generation of novel influenza viruses capable of crossing the species barrier to humans. There are three subtypes of SI viruses (SIV) currently circulating in pigs in the U.S.: H1N1, H3N2, and H1N2 (Olsen, 2002, Virus Res 85:199-210; Karasin et al., 2002, J Clin Microbiol 40:1073-9; Karasin et al., 2000, Virus Res 68:71-85; Olsen et al., 2000, Arch Virol 145:1399-419; Webby et al., 2000, J Virol 74:8243-51; Webby et al., 2001, Philos Trans R Soc Lond 20 B Biol Sci 356:1817-28; Zhou, 2000, Vet Microbiol 74:47-58). Before 1998, mainly "classical" H1N1 SIVs were isolated from swine in the United States (Kida et al., 1994, J Gen Virol 75:2183-8; Scholtissek, 1994, Eur J Epidemiol 10:455-8; Olsen et al., 2000, Arch Virol. 145:1399-419). In 1998, SIVs of the subtype H3N2 were isolated in multiple states in the United States. These viruses were generated by reassortment between human, avian and classical swine viruses, they are undergoing rapid evolution and in general they cause more severe disease than classical H1N1 SIV. 25 [0009] Pathogenicity of influenza viruses is modulated by multiple virus and host factors. Among the host factors that fight virus infections, the type I interferon (IFNα/β) system represents a powerful antiviral innate defense mechanism which was established relatively early in the evolution of eukaryotic organisms (Garcia-Sastre, 2002, Microbes Infect 4:647-55). The antiviral IFNα/β system involves three major steps: (i) detection of viral infection and IFNα/β secretion, (ii) binding of IFNα/β to its receptors and transcriptional induction of IFNα/β-stimulated genes, and (iii) synthesis of 30 antiviral enzymes and proteins. Most viruses, however, have acquired specific genetic information encoding IFNα/β antagonist molecules, which effectively block one or more steps of the antiviral IFNα/β system. Influenza A viruses express a non-structural protein in infected cells, the NS1 protein, which counteracts the cellular IFN α/β response (Garcia- Sastre et al., 1998, Virology 252:324-30). [0010] Influenza infection in pigs was first reported in 1918 and the first swine influenza viruses were isolated from 35 pigs in 1930 (Shope, R.E., 1931, J. Exp. Med. 54:373-385). These first isolates were the progenitors of what is recognized as the H1N1 lineage of swine influenza A viruses. From 1930 to the 1990s, influenza in North American pigs was caused almost exclusively by infection with H1N1 swine viruses. A dramatic shift in the pattern of swine influenza began around 1997, when an unexpected and substantial increase in H3 seropositivity (8%) was detected, and H3N2 viruses began to be isolated from pigs in both the US and Canada (Olsen et al., 2000, Arch. Virol. 134:1399-1419). Furthermore, 40 reassortment between H3N2 viruses and H1N1 swine viruses resulted in the detection of second generation H1N2 reassortant viruses (Karasin et al., 2000, J. Clin. Microbiol. 38:2453-2456; Karasin et al., 2002, J. Clin Microbiol. 40:1073-1079). In addition, avian H4N6 viruses of duck origin have been isolated from pigs in Canada (Karasin et al., 2000, J. Virol. 74:9322-9327). The generation of these novel viruses in addition to the described antigenic drift variants of H1N1 swine influenza viruses, introduce potential veterinary and human public health implications. 45 [0011] In 1998, a new strain of swine influenza virus to which pigs had little immunity sickened every pig in an operation of 2400 animals. Although there has been only one influenza subtype which has sickened North American pigs since 1930, in the last few years a quick succession of new flu viruses has been sweeping through North America’s 100 million pigs. After years of stability, the North American swine flu virus has jumped onto an evolutionary fast track, bringing out variants every year. This has had not only an undesired effect on the fanning industry and a negative economic impact, 50 but, there is also concern by experts that the evolving swine flu increases the likelihood that a novel virus will arise that is transmissible among humans. Fortunately, the new pig strains that have appeared in North America so far do not appear to readily infect humans.

2.3 Attenuated Viruses 55 [0012] Inactivated virus vaccines are prepared by "killing" the viral pathogen, e.g., by heat or formalin treatment, so that it is not capable of replication. Inactivated vaccines have limited utility because they do not provide long lasting immunity and, therefore, afford limited protection. An alternative approach for producing virus vaccines involves the use

5 EP 2 497 492 B1

of attenuated live virus vaccines. Attenuated viruses are capable of replication but are not pathogenic, and, therefore, provide for longer lasting immunity and afford greater protection. However, the conventional methods for producing attenuated viruses involve the chance isolation of host range mutants, many of which are temperature sensitive; e.g., the virus is passaged through unnatural hosts, and progeny viruses which are immunogenic, yet not pathogenic, are 5 selected. [0013] A conventional substrate for isolating and growing influenza viruses for vaccine purposes are embryonated chicken eggs. Influenza viruses are typically grown during 2-4 days at 37°C in 10-12 day old eggs. Although most of the human primary isolates of influenza A and B viruses grow better in the amniotic sac of the embryos, after 2 to 3 passages the viruses become adapted to grow in the cells of the allantoic cavity, which is accessible from the outside of the egg 10 (Murphy, B.R., and R.G. Webster, 1996. Orthomyxoviruses pp. 1397-1445. In Fields Virology. Lippincott-Raven P.A.). [0014] Recombinant DNA technology and genetic engineering techniques, in theory, would afford a superior approach to producingan attenuated virussince specific mutations could be deliberately engineeredinto the viral genome. However, the genetic alterations required for attenuation of viruses are not known or predictable. In general, the attempts to use recombinant DNA technology to engineer viral vaccines have mostly been directed to the production of subunit vaccines 15 which contain only the protein subunits of the pathogen involved in the immune response, expressed in recombinant viral vectors such as vaccinia virus or baculovirus. More recently, recombinant DNA techniques have been utilized in an attempt to produce herpes virus deletion mutants or polioviruses which mimic attenuated viruses found in nature or known host range mutants. Until 1990, the negative strand RNA viruses were not amenable to site-specific manipulation at all, and thus could not be genetically engineered. 20 [0015] Although these viruses are beneficial because they are immunogenic and not pathogenic, they are difficult to propagate in conventional substrates for the purposes of making vaccines. Furthermore, attenuated viruses may possess virulence characteristics that are so mild as to not allow the host to mount an immune response sufficient to meet subsequent challenges. [0016] Human influenza viruses does not replicate efficiently in birds, and vice versa due to differences in receptors 25 which bind the viruses. In contrast, pigs are uniquely susceptible to infection with human and avian viruses because they possess receptor types present both in humans and avian influenza viruses. As a result, pigs have been hypothesized to be the "mixing vessel" hosts for human-avian virus reassortment and there is support for this theory from several studies. See, e.g., Shu et al., 1994, Virology 202:825-33; Scholtissek, 1990, Med. Principles Pract. 2:65-71; Zhou et al., 1999 J Virol. 73:8851-6. This mixing facilitates the generation of novel human influenza virus strains and the initiation 30 of influenza pandemics. [0017] Inactivated or "killed" influenza virus preparations are the only influenza vaccines currently licensed in the United States. An alternative approach for producing virus vaccines to the inactivated virus vaccines in which the viral pathogen is "killed", involves the use of attenuated live virus vaccines which are capable of replication but are not pathogenic. Live vaccines which are administered intranasally may have advantages over their inactivated counterparts. 35 Firstly, live vaccines are thought to induce improved cross-reactive cell-mediated cytotoxicity as well as a humoral antibody response, providing better protection than inactivated vaccines (Gorse and Belshe, 1990, J. Clin. Microbiol. 28:2539-2550; and Gorse et al., 1995, J. Infect. Dis. 172:1-10). Secondly, live vaccines also have the advantage of intranasal administration which avoids the swelling and muscle soreness occasionally associated with the intramuscular administration of inactivated adjuvanted vaccines. These live vaccines have been reported to induce not only humoral 40 responses against homotypic influenza virus but also crossreactive cell-mediated cytotoxicity. Futher advantages of live vaccines include the ease of intranasal administration, induction of mucosal immunity, longer lasting immunity, and its cost effectiveness. These are all important considerations regarding potential swine influenza vaccines. [0018] Thus, new and more effective vaccines and immunogenic formulations for preventing swine influenza virus infections generated by such technology are needed. 45 3. SUMMARY OF THE INVENTION

[0019] The present invention provides a genetically engineered attenuated swine influenza virus having an altered interferon antagonist phenotype, wherein said virus comprises a swine influenza virus A/Swine/Texas/4199-2/98 NS1 50 gene with a mutation resulting in an NS1 protein as set out in claim 1. Other embodiments of the invention are set out in claims 2-27. [0020] The present invention further provides an immunogenic formulation comprising the genetically engineered attenuated swine influenza virus, and a physiologically acceptable excipient. Also, the present invention provides a pharmaceutical formulation comprising the genetically engineered attenuated swine influenza virus of the present in- 55 vention and a pharmaceutically acceptable carrier. [0021] Furthermore, the present invention provides the use of an effective amount of the immunogenic formulation of the present invention in the preparation of a medicament for use in immunizing or inducing an immune response in a pig. Also, the present invention provides for use of an effective amount of the genetically engineered attenuated swine

6 EP 2 497 492 B1

influenza virus of the present invention in the preparation of a medicament, wherein the medicament is (a) for use in the prevention of a condition or a symptom associated with swine influenza virus in a pig; or (b) for use in the treatment of a swine influenza virus infection in a pig. [0022] The present invention also provides a method for production of an immunogenic formulation comprising (a) 5 propagating in a substrate the genetically engineered attenuated swine influenza virus of the present invention and (b) collecting progeny virus, wherein the substrate is a cell, cell line or embryonated egg. [0023] Furthermore, the present invention provides a cultured cell containing the genetically engineered attenuated swine influenza virus of the present invention. The present invention also provides an embryonated egg containing the genetically engineered attenuated swine influenza virus of the present invention. 10 [0024] Disclosed herein are attenuated swine influenza viruses having an impaired ability to antagonize the cellular interferon (IFN) response, methods for producing such attenuated swine influenza viruses, and the use of such viruses in vaccine and pharmaceutical formulations. Such viruses are capable of generating an immune response and creating immunitybut not causingillness or causingfewer and/or less severe symptoms, i.e., the viruses have decreased virulence. Therefore, they are ideal candidates for live virus vaccines. Moreover, the attenuated viruses can induce a robust IFN 15 response which has other biological consequences in vivo, affording protection against subsequent infectious diseases and/or inducing antitumor responses. Therefore, the attenuated viruses can be used pharmaceutically, for the prevention or treatment of other infectious diseases and/or IFN-treatable diseases. [0025] The invention is based, in part, on the Applicants’ discovery that swine influenza viruses engineered to contain or containing a deletion(s) in the NS1 gene have impaired replication relative to wild-type swine influenza viruses as 20 demonstrated by fewer lung lesions upon infection of pigs, reduced viral titers in bronchoalveolar lavage fluid (BALF) and reduced detection of virus on nasal swabs. Surprisingly, and contrary to results seen with human influenza virus in mouse models, the length of the NS1 protein does not correlate with the level of attenuation of the swine influenza virus. Applicants have discovered that with swine influenza, a mutant virus with the shortest NS1 protein is the least attenuated. In other words, swine influenza viruses containing shorter deletions in the NS1 gene exhibit a greater attenuation in vivo 25 than swine influenza viruses containing longer deletions in their NS1 gene, or wild type swine influenza viruses. Applicants have further discovered that the recombinant swine influenza viruses are impaired in their ability to inhibit IFN production in vitro, and they do not replicate as efficiently as the parental recombinant strain in 10-day old embryonated hen eggs, in MDCK cells, in PK-15 cells or in an in vivo pig model. While not intending to be bound to any theory or explanation for the mechanism of action of the swine influenza virus NS1 deletion mutants in vivo, the attenuated features of such 30 viruses are presumably due to their levels of NS1 protein expression, their ability to induce a robust cellular IFN response, and their impaired ability to antagonize such a response. However, the beneficial features of such viruses may not be solely attributable to their effect on the cellular interferon response. Indeed, alterations in other activities associated wit h NS1, such as, alteration of pre-mRNA splicing, inhibition of cellular mRNA polyadenylation, poly(A)-containing mRNA nucleocytoplasmic transport, and stimulation of viral protein synthesis, may contribute to the desired attenuated pheno- 35 type achieved by the introduction of mutations in the NS1 gene of swine influenza virus. [0026] An attenuated swine influenza virus disclosed herein comprises a mutation in a swine influenza NS1 gene that diminishes the ability of the NS 1 gene product to antagonize the cellular interferon response. An attenuated swine influenza virus disclosed herein may comprise a genome comprising a mutation in a swine influenza virus NS1 gene that diminishes the ability of the NS1 gene product to antagonize a cellular interferon response, and permits the attenuated 40 virus, at a multiplicity of infection (MOI) of between 0.0005 and 0.001, 0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or a MOI of 0.0005, 0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately 1 to approximately 100 fold, approximately 5 to approximately 80 fold, approximately 20 to approximately 80 fold, or approximately 40 to approximately 80 fold, approximately 1 to approximately 10 fold, approximately 1 to approximately 5 fold, approximately 1 to approximately 4 fold, approximately 1 to approximately 3 fold, 45 approximately 1 to approximately 2 fold, approximately 3 to approximately 15 fold, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold lower than wild-type swine influenza virus in cells (e.g., cells of a human(e.g., PerC6, a producer cell line derived from human embryonic retinoblasts transformed with the E1 region of Adenovirus 5), mouse, chicken (e.g., chicken embryo fibroblasts), rat, birds, or pig (e.g., PK(D1) cells, PK(15) cells, PK13 cells, NSK cells, LLC-PK1 cells, LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 50 cells, PK-2a/CL 13 or SJPL cells)), as determined approximately 2 to 10 days, 3 to 7 days, 3 to 5 days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 days post-infection when propagated under the same conditions. The titers of attenuated and wild-type swine influenza viruses can be determined utilizing any technique well-known in the art or described herein, (e.g., hemagglutination assays, plaque assays, tissue culture infectious dose 50 (TCID50), egg infectious dose 50 (EID50), etc.) and the viruses can be propagated under conditions described herein or well-known in the art (e.g., in pig cells, MDCK cells 55 (e.g., in MEM, 10% v/v fetal calf serum (FCS), 1% penicillin/streptomycin at 37°C in a 5% CO 2 humidified incubator) or embryonated chicken eggs (e.g., in a stationary incubator at 37°C with 55% relative humidity). Alternatively, the viruses can be propagated in cells (e.g., in pig cells, MDCK cells, etc.) that are grown in serum-free or serum reduced(e.g., TPCK trypsin) medium.

7 EP 2 497 492 B1

[0027] In a specific aspect, an attenuated swine influenza virus disclosed herein comprises a genome comprising a mutation in a swine influenza virus NS1 gene that diminishes the ab ility of the NS1 g ene product to antagonize a cellular interferon response, and permits the attenuated virus, at a multiplicity of infection (MOI) of between 0.0005 and 0.001, 0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or a MOI of 0.0005, 0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 5 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately 1 to approximately 100 fold, approximately 5 to approximately 80 fold, approximately 20 to approximately 80 fold, or approximately 40 to approximately 80 fold, approximately 1 to approximately 10 fold, approximately 1 to approximately 5 fold, approximately 1 to approximately 4 fold, approximately 1 to approximately 3 fold, approximately 1 to approximately 2 fold, approximately 3 to approximately 15 fold, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 10 fold lower than wild-type swine influenza virus in cells (e.g., cells of a human (e.g., PerC6, a producer cell line derived from human embryonic retinoblasts transformed with the E1 region of Adenovirus 5), mouse, chicken(e.g., chicken embryo fibroblasts), rat, birds, or pig (e.g., PK(D1) cells, PK(15) cells, PK13 cells, NSK cells, LLC-PK1 cells, LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells, PK-2a/CL 13 or SJPL cells)), as determined approximately 2 to 10 days, 3 to 7 days; 3 to 5 days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 days post-infection when propagated under the same conditions. The 15 titers of attenuated and wild-type swine influenza viruses can be determined utilizing any technique well-known in the art or described herein, (e.g., hemagglutination assays, plaque assays, etc.) and the viruses can be propagated under conditions described herein or well-known in the art (e.g., in pig cells, MDCK cells (e.g., in MEM, 10% v/v fetal calf serum

(FCS), 1% penicillin/ streptomycin at 37°C in a 5% CO2 humidified incubator) or embryonated chicken eggs (e.g., in a stationary incubator at 37°C with 55% relative humidity). 20 [0028] The swine influenza viruses used in accordance with the present disclosure may be selected from naturally occurring strains, variants or mutants; mutagenized viruses (e.g., viruses generated by exposure to mutagens, repeated passages and/or passage in non-permissive hosts); reassortants; and/or genetically engineered viruses (e.g., using the "reverse genetics" and helper-free plasmid-based techniques) having the desired phenotype - i.e., an impaired ability to antagonize the cellular IFN response. The naturally occurring strains, variants or mutants, reassortments and/or 25 genetically engineered viruses with the desired interferon antagonist phenotype can be selected based on differential growth in cells (e.g., cells of a human (e.g., PerC6, a producer cell line derived from human embryonic retinoblasts transformed with the E1 region of Adenovirus 5), mouse, chicken (e.g., chicken embryo fibroblasts), rat, birds, or pig (e.g., PK(D1) cells, PK(15) cells, PK13 cells, NSK cells, LLC-PK1 cells, LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells, PK-2a/CL 13 or SJPL cells)) in other assays described below. The swine influenza viruses of the invention may 30 be genetically engineered viruses. The swine influenza viruses of the invention may be non naturally occuring strains, variants or mutants and/or reassortments. [0029] In a specific aspect, an attenuated swine influenza virus disclosed herein comprises a genome comprising a mutation in a swine influenza virus NS1 gene that diminishes the ab ility of the NS1 g ene product to antagonize a cellular interferon response, and permits the attenuated virus, at a multiplicity of infection (MOI) of between 0.0005 and 0.001, 35 0.001 and 0.01,0.01 and 0.1, or 0,1 and 1, or a MOI of 0.0005, 0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately 1 to approximately 100 fold, approximately 5 to approximately 80 fold, approximately 20 to approximately 80 fold, or approximately 40 to approximately 80 fold, approximately 1 to approximately 10 fold, approximately 1 to approximately 5 fold, approximately 1 to approximately 4 fold, approximately 1 to approximately 3 fold, approximately 1 to approximately 2 fold, approximately 3 to approximately 40 15 fold, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold lower than wild-type swine influenza virus in cells (e.g., cells of a human (e.g., PerC6, a producer cell line derived from human embryonic retinoblasts transformed with the E1 region of Adenovirus 5), mouse, chicken(e.g., chicken embryo fibroblasts), rat, birds, or pig (e.g., PK(D1) cells, PK(15) cells, PK13 cells, NSK cells, LLC-PK1 cells, LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells, PK-2a/CL 13 or SJPL cells)) as determined by a hemagglutination assay of 45 BALF from pigs or supernatants of pig cells approximately 2 to 10 days, 3 to 7 days, 3. to 5 days, or 2, 3, 5, 6, 7, 8, 9, 10 days post-infection or when the viruses are plaqued on Madin-Darby canine kidney (MDCK) cells. The growth of an attenuated swine influenza virus of the invention may be compared to a particular standard or reference, e.g., wild-type swine influenza virus A/Swine/Texas/4199-2/98. The attenuated virus may be genetically-engineered to contain or express non-swine influenza virus nucleic acid sequences such, e.g., an epitope of a foreign pathogen or a tumor antigen. 50 Preferably, the non-swine influenza virus sequences do not include a nucleic acid sequence that alters the attenuated phenotype of the virus. Accordingly, nucleic acid sequences encoding proteins, polypeptides or peptides with interferon antagonizing activity are preferably not engineered into a swine influenza virus. [0030] Disclosed herein are attenuated swine influenza viruses comprising a genome comprising at least two, at least three, at least four or more mutations in two, three, four or more swine influenza virus genes, wherein at least one of 55 the mutations is in the NS1 gene and contributes to or is responsible (directly or indirectly) for the attenuation of the virus and/or the diminished ability of the virus to antagonize a cellular interferon response. In one aspect, an attenuated swine influenza virus disclosed herein comprises a genome comprising at least two, at least three, at least four or more mutations in two, three, four or more swine influenza virus genes, wherein at least one of the mutations is in the NS 1

8 EP 2 497 492 B1

gene and is responsible for the diminished ability of the NS1 gene product to antagonize a cellular interferon response, and permits the attenuated virus, at a MOI of between 0.0005 and 0.001,0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or a MOI of 0.0005, 0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately 1 to approximately 100 fold, approximately 5 to approximately 80 fold, approximately 5 20 to approximately 80 fold, or approximately 40 to approximately 80 fold, approximately 1 to approximately 10 fold, approximately 1 to approximately 5 fold, approximately 1 to approximately 4 fold, approximately 1 to approximately 3 fold, approximately 1 to approximately 2 fold, approximately 3 to approximately 15 fold or approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold lower than wild-type swine influenza virus in cells (e.g., cells of a human(e.g., PerC6, a producer cell line derived from human embryonic retinoblasts 10 transformed with the E1 region of Adenovirus 5), mouse, chicken (e.g., chicken embryo fibroblasts), rat, birds, or pig (e.g., PK(D1) cells, PK(15) cells, PK13 cells, NSK cells, LLC-PK1 cells, LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells, PK-2a/CL 13 or SJPL cells)), as determined by, e.g., hemagglutination assays, approximately 2 to 10 days, 3 to 7 days, 3 to 5 days, or 2,3,5,6,7,8,9,10 days post-infection when the viruses are propagated under the same conditions. [0031] In a specific aspect, an attenuated swine influenza virus disclosed herein comprises a genome comprising at 15 least two, at least three, at least four or more mutations in two, three, four or more swine influenza virus genes, wherein at least one of the mutations is in the NS1 gene and is responsible for the diminished ability of the NS1 gene product to antagonize a cellular interferon response, and permits the attenuated virus, at a MOI of between 0.0005 and 0.001, 0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or a MOI of 0.0005, 0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately 1 to approximately 100 fold, approximately 20 5 to approximately 80 fold, approximately 20 to approximately 80 fold, or approximately 40 to approximately 80 fold, approximately 1 to approximately 10 fold, approximately 1 to approximately 5 fold, approximately 1 to approximately 4 fold, approximately 1 to approximately 3 fold, approximately 1 to approximately 2 fold, approximately 3 to approximately 15 fold or approximately 1,2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold lower than wild-type swine influenza virus in pig cells, as determined by, e.g., hemagglutination assays, approximately 25 2 to 10 days, 3 to 7 days, 3 to 5 days, or 2, 3, 5, 6, 7, 8, 9,10 days post-infection when the viruses are propagated under the same conditions (e.g., in MDCK cells). In another aspect, an attenuated swine influenza virus disclosed herein comprises a genome comprising at least two, three, four or more mutations in two, three, four or more swine influenza virus genes, wherein at least one of the mutations is in the NS1 gene and is responsible for the attenuation of the virus, and permits the attenuated virus, at a MOI of between 0.0005 and 0.001, 0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or 30 a MOI of 0.0005, 0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately I to approximately 100 fold, approximately 5 to approximately 80 fold, approximately 20 to approximately 80 fold, or approximately 40 to approximately 80 fold, approximately 1 to approximately 10 fold, approximately 1 to approximately 5 fold, approximately 1 to approximately 4 fold, approximately 1 to approximately 3 fold, approximately 1 to approximately 2 fold, approximately 3 to approximately 15 fold or approximately 1, 2, 3, 4, 5, 6, 7, 8, 35 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold lower than wild-type swine influenza virus in pig cells, as determined by, e.g., hemagglutination assays, approximately 2 to 10 days, 3 to 7 days, 3 to 5 days, or 2, 3, 5, 6, 7, 8, 9, 10 days post-infection when the viruses are propagated under the same conditions(e.g ., in MDCK cells). In accordance with these aspects, the attenuated virus has an impaired interferon antagonist phenotype and may be genetically engineered to contain or express non-swine influenza virus nucleic acid sequences, such as,e.g ., an 40 epitope of a foreign pathogen (e.g., epitopes of porcine reproductive and respiratory syndrome virus, porcine cytomegalo virus, porcine respiratory corona virus, porcine encephalomyocarditis virus, porcine epidemic diarrhea and antigenic determinants of non-viral swine pathogens such as bacteria, including, but not limited to, Brucella suis, and parasites, including, but not limited to, roundworms (Ascaris suum), whipworms (Trichuris suis), or a tumor antigen such as carcinoembryonic antigen (CEA), breast cancer antigen such as EGFR (epidermal growth factor receptor), HER2 antigen 45 (p185HER2), HER2 neu epitope, cancer antigen-50 (CA-50), cancer antigen 15-3 (CA15-3) associated with breast cancer, carcinoma associated antigen (CAA), melanoma antigen, and melanoma associated antigens 100, 25, and 150). Pref- erably, the non-swine influenza virus sequences (heterologous sequences) do not include a nucleic acid sequence that alters the attenuated phenotype of the virus. Accordingly, nucleic acid sequences encoding proteins, polypeptides or peptides with interferon antagonizing activity are preferably not engineered into the swine influenza virus. 50 [0032] Mutations in the swine influenza virus NS1 gene comprise (alternatively, consist of) any mutation that results in the desired phenotype (i.e., an impaired ability to antagonize a cellular interferon response). Examples of the type of mutations that can be included in or introduced into a swine influenza virus NS1 gene include, but are not limited to, deletions, substitutions, insertions and combinations thereof. One or more mutations can be located anywhere throughout the NS1 gene, i.e., in the regulatory, non-coding and/or coding regions (e.g., the amino-terminus and/or the carboxy- 55 terminus or somewhere in between). In a specific aspect, an attenuated swine influenza virus disclosed herein comprises a genome comprising a swine influenza virus NS1 gene with a mutation e.g.,( a deletion or substitution) at the amino terminus. In a preferred aspect, an attenuated swine influenza virus disclosed herein comprises a genome comprising a swine influenza NS1 gene with a mutation ( e.g., a deletion or substitution, preferably a deletion) at the carboxy terminus.

9 EP 2 497 492 B1

In another aspect, an attenuated swine influenza virus disclosed herein comprises a genome comprising a mutation in a swine influenza virus NS 1 gene resulting in a deletion consisting of 5, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 100, 105, 110, 115, 119, 120, 121, 125, 130, 135, 140, 145, 146, 147, 148, 150, 155, 160, 165, 170 or 175 amino acid residues from the carboxy terminus of NS1, or a deletion of between 5 5-170, 25-170, 50-170, 100-170, 90-160, 100-160 or 105-160, 90-150, 5-75, 5-50 or 5-25 amino acid residues from the carboxy terminus. In another aspect, an attenuated swine influenza virus disclosed herein comprises a genome comprising a mutation in a swine influenza virus NS1 gene resulting in a deletion of all amino acid residues of the NS1 gene product except amino acid residues 1-126, amino acid residues 1-120, amino acid residues 1-115, amino acid residues 1-110, amino acid residues 1-100, amino acid residues 1-99, amino acid residues 1-95, amino acid residues 1-85, amino 10 acid residues 1-80, amino acid residues 1-75, amino acid residues 1-73, amino acid residues 1-70, amino acid residues 1-65 or amino acid residues 1-60, wherein the amino terminal amino acid is number 1. The attenuated swine influenza virus may be genetically engineered. An attenuated swine influenza virus of the disclosure is TX/98/del 126, TX/98/del 99 or TX/98/del 73. [0033] The attenuated swine influenza virus of the present invention may be a chimeric virus that expresses a heter- 15 ologous sequence. Preferably, the heterologous sequence is not a nucleic acid sequence that alters the attenuated phenotype of the virus. Accordingly, preferably, nucleic acid sequences encoding proteins, polypeptides or peptides with interferon antagonizing activity are not engineered into the swine influenza virus. The chimeric virus may express a tumor antigen. The chimeric virus may express an epitope of a foreign pathogen. [0034] An attenuated swine influenza virus having the desired phenotype can itself be used as the active ingredient 20 in vaccine, pharmaceutical or immunogenic formulations. Alternatively, the attenuated swine influenza virus can be used as the vector or "backbone" of recombinantly produced vaccines or immunogenic formulations. To this end, the "reverse genetics" technique or helper-free plasmid approach can be used to engineer mutations or introduce foreign epitopes into the attenuated swine influenza virus, which would serve as the "parental" strain. In this way, vaccines can be designed for immunization against strain variants, or in the alternative, against completely different infectious agents or disease 25 antigens (e.g., tumor antigens). For example, attenuated swine influenza virus can be engineered to express neutralizing epitopes of other preselected strains. Alternatively, epitopes of other viruses can be built into the attenuated swine influenza virus. Alternatively, epitopes of non-viral infectious pathogens (e.g., parasites, bacteria, fungi) can be engineered into the swine influenza virus. [0035] Reassortment techniques can be used to transfer the attenuated phenotype from a parental swine influenze 30 virus strain (a natural mutant, a mutagenized virus, or a genetically engineered virus) to a different virus strain (a wild- type virus, a natural mutant, a mutagenized virus, or a genetically engineered virus). The "reverse genetics technique" or helper-free plasmid approach can be used to engineer mutations or introduce foreign epitopes into the attenuated swine influenza virus, which would serve as the "parental strain". [0036] Diclosed herein are methods for vaccinating subjects comprising administering a vaccine formulation comprising 35 an attenuated swine influenza virus comprising a mutation in a swine influenza NS1 gene that diminishes the ability of the NS1 gene product to antagonize the cellular interferon response, and a physiologically effective excipient. In one aspect, disclosed herein is a method comprising administering an effective amount of a vaccine formulation disclosed herein. In certain aspects, the dose of the vaccine formulation administered is between about 102 to about 108, about 103 to about 107, or about 104 to about 5 x 106 pfu. In other aspects, the dose of the vaccine formulation administered 40 is 102, 5 x 102, 103, 5 x 103, 104, 5 x 104, 105, 5 x 105, 106, 5 x 106, 107, 5 x 107 or 108 pfu. In specific aspects, the subject is a donkey, zebra, camel, dog, avian (e.g., a duck). In a preferred aspect, the subject is a pig. [0037] The present invention provides immunogenic formulations comprising an attenuated swine influenza virus of the invention. Disclosed herein are methods for inducing an immune response for the treatment, management or prevention of a swine influenza virus infection or a condition or symptom associated therewith, an infection, other than a 45 swine influenza virus infection, or a condition or symptom associated therewith, or a condition in which the attenuated swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition, comprising administering to a subject an immunogenic formulation of the invention. In one aspect, the present invention relates to an immunogenic formulation for use in a method for immunizing or inducing an immune response e.g., for the treatment, management or prevention of a swine influenza virus infection or a condition or symptom 50 associated therewith, comprising administering to a subject an effective amount of an immunogenic formulation of the invention. The dose of immunogenic formulation of the invention to be administered to a subject may be between about 102 to about 10 8, about 10 3 to about 107, or about 10 4 to about 5 x 10 6 pfu or about 10 4 to about 10 7 pfu. The immunogenic formulation to be administered to a subject may have an attenuated swine influenza virus of the present invention at a concentration of about 10 4 to about 10 7 pfu/ml. The dose of immunogenic formulation of the invention to be administered 55 to a subject may be 10 2, 5 x 102, 103, 5 x 10 3, 104, 5 x 104, 105, 5 x 10 5, 106, 5 x 10 6, 107, 5 x 10 7 or 108 pfu. The subject may be a donkey, zebra, camel, dog, avian (e.g., a duck), and especially a pig. [0038] The attenuated swine influenza viruses, which induce robust IFN responses in subjects, may also be used in pharmaceutical formulations for the prophylaxis or treatment of infections and IFN-treatable diseases such as cancer.

10 EP 2 497 492 B1

In this regard, the tropism of the attenuated swine influenza virus can be altered to target the virus to a desired target organ, tissue or cells in vivo or ex vivo. Using this approach, the IFN response can be induced locally, at the target site, thus avoiding or minimizing the side effects of systemic IFN therapy. To this end, the attenuated swine influenza virus can be engineered to express a ligand specific for a receptor of the target organ, tissue or cells. 5 [0039] Disclosed herein are methods for preventing, managing or treating an infection, or IFN-treatable disease in a subject (e.g., a pig), other than a swine influenza viral infection, or IFN-treatable disease caused by swine influenza virus, comprising administering a pharmaceutical formulation disclosed herein. In one aspect, disclosed herein is a method for preventing, managing or treating an infection or IFN-treatable disease in a subject, other than a swine influenza viral infection or IFN-treatable disease caused by swine influenza virus, comprising administering to a subject 10 an effective amount of a pharmaceutical formulation disclosed herein. In certain embodiments, The dose of the pharmaceutical formulation to be administered to the subject may be between about 102 to about 1012, about 102 to about 1010, about 102 to about 10 8, about 10 3 to about 10 9, about 103 to about 10 7, about 104 to about 10 8, about 10 4 to about 5 x 106, about 104 to about 107, or about 104 to about 1012 pfu. The pharmaceutical formulation to be administered to the subject may have an attenuated influenza virus of the present invention at a concentration from about 10 4 to about 15 1012 pfu/ml. The dose of the pharmaceutical formulation to be administered may be 10 2, 5 x 102, 103, 5 x 103, 104, 5 x 104, 105, 5 x 105, 106, 5x 106, 107, 5 x 107, 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011, 5 x 1011 or 1012 pfu. The subject may be a donkey, zebra, camel, dog, avian ( e.g., a duck) and especially a pig. [0040] Disclosed herein are methods for preventing, managing or treating cancer in a subject comprising administering a pharmaceutical formulation disclosed herein. In one aspect, disclosed herein is a method for preventing, managing 20 or treating cancer in a subject comprising administering to a subject an effective amount of a pharmaceutical formulation disclosed herein. The pharmaceutical formulation may comprise a swine influenza virus comprising a tumor antigen. The dose of the pharmaceutical formulation to be administered to the subject may be between about 10 2 to about 10 12, about 102 to about 1010, about 102 to about 108, about 103 to about 109, about 103 to about 107, about 104 to about 108, about 104 to about 5 x 106 pfu or about 104 to about 1012 pfu. The dose of the pharmaceutical formulation to be 25 administered may be 102, 5 x 102, 103, 5 x 103, 104, 5 x 104, 105, 5 x 105, 106, 5 x 106, 107, 5 x 107, 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011, 5 x 1011 or 1012 pfu. The subject may be a donkey, zebra, camel, dog, avian (e.g., a duck) and especially a pig. [0041] Applicants have demonstrated that attenuated swine influenza viruses with impaired interferon antagonist activity replicate in vivo generating titers that are lower than than detected with wild-type swine influenza viruses, but 30 that are sufficient to induce immunological and cytokine responses. Thus, mutations which diminish but do not abolish the IFN antagonist activity of the swine influenza virus are preferred for vaccine, immunologic, and pharmaceutical formulations. Such viruses can be selected for growth in both conventional and non-conventional substrates, and for intermediate virulence. [0042] The present invention provides cells containing (alternatively, comprising) swine influenza viruses of the present 35 invention. A cell may contain comprise a swine influenza virus that is genetically engineered. The attenuated swine influenza virus contained in the cell may be engineered to encode an epitope derived from another pathogene.g (., another virus) or a tumor antigen. The genome of the attenuated swine influenza virus may comprise at least one segment derived from a different virus. Any cell can be infected with, contain or comprise an attenuated swine influenza virus of the invention including, but not limited to, MDCK cells, PK cells, Vero cells, primary human umbilical vein endothelial 40 cells (HUVEC), H292 human epithelial cell line, HeLa cells, and swine embryonic kidney cells RES. The cell may be a pig cell or a pig cell line. The cell ( e.g., a pig cell or pig cell line) may be interferon deficient. [0043] The swine influenza viruses of the invention may be contained in 10 day-old, 6 to 9 day-old, 6 to 8-day-old, or 6 to 7 day-old embryonated chicken eggs. The viruses may be contained in allantoic cavity of such eggs. The viruses may be contained in the amniotic cavity of such eggs. 45 [0044] The invention encompasses the use of substrates such as cells, cell lines and embryonated eggs, to propagate the attenuated swine influenza viruses of the invention. In one embodiment, the invention provides methods for vaccine production immunogenic formulation production and pharmaceutical production comprising propagating in a substrate an attenuated swine influenza virus of the present invention and collecting progeny virus, wherein the substrate is a cell, cell line or embryonated egg. In certain embodiments, the substrate is an IFN deficient system. Exemplary IFN-deficient 50 systems include, but are not limited to, young embryonated eggs (e.g., 6 to 10 days old, 6 to 9 days old, 6 to 8 days old or 6 to 7 days old embryonated eggs), and IFN-deficient cell lines (such as VERO cells or genetically engineered cell lines such as STAT1 knockouts). Embryonated eggs or cell lines pretreated with compounds that inhibit the IFN system (including drugs, antibodies, antisense, ribozymes, etc.) can also be used as an IFN-deficient system. Further, eggs deficient in the IFN system, e.g., eggs produced by STAT1 negative birds, especially fowl, including but not limited to 55 transgenic chickens, ducks or turkeys, may be used as an IFN-deficient system.

11 EP 2 497 492 B1

3.1 Terminology

[0045] As used herein, the term "about" or "approximately" when used in conjunction with a number refers to any number within 1, 5 or 10% of the referenced number. 5 [0046] As used herein, the phrase "amino-terminus" of NS1 refer to the amino acids from the amino terminal amino acid residue (amino acid residue 1) through amino acid residue 1.15, amino acid residues 1 through 100, amino acid residues 1 through 75, amino acid residues 1 through 50, amino acid residues 1 through 25, or amino acid residues 1 through 10 of the swine influenza viral NS1 protein. [0047] As used herein, the phrase "carboxy-terminus" of NS1 refer to amino acid residues 116 through the carboxy 10 terminal amino acid residue, amino acid residues 101 through the carboxy terminal amino acid residue, amino acid residues 76 through the carboxy terminal amino acid residue, amino acid residues 51 through the carboxy terminal amino acid residue, or amino acid residues 26 through the carboxy terminal amino acid residue of the swine influenza viral NS1 protein, when the amino-terminus of NS1 is amino acid residues 1 through amino acid residue 115, amino acid residues 1 through 100, amino acid residues 1 through 75, amino acid residues 1 through 50, or amino acid residues 15 1 through 25, respectively, of the swine influenza viral NS1 protein. Deletions from the carboxy terminus can include deletions consisting of 5, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 73, 75, 80, 85, 90, 95, 99, 100, 105,110, 115, 120, 125, 126, 130, 135, 140, 145, 150, 155, 160, 165, 170 or 175 amino acid residues from the carboxy terminus of NS1. [0048] As used herein, the term "cytokine receptor modulator" refers to an agent which modulates the phosphorylation 20 of a cytokine receptor, the activation of a signal transduction pathway associated with a cytokine receptor, and/or the expression ofa particularprotein such as a cytokine.Such anagent may directly orindirectly modulatethe phosphorylation of a cytokine receptor, the activation of a signal transduction pathway associated with a cytokine receptor, and/or the expression of a particular protein such as a cytokine. Thus, examples of cytokine receptor modulators include, but are not limited to, cytokines, fragments of cytokines, fusion proteins, and antibodies that immunospecifically binds to a 25 cytokine receptor or a fragment thereof. Further, examples of cytokine receptor modulators include, but are not limited to, peptides, polypeptides ( e.g., soluble cytokine receptors), fusion proteins and antibodies that immunospecifically binds to a cytokine or a fragment thereof. [0049] As used herein, the term "effective amount" refers to the amount of a therapy ( e.g., a prophylactic or therapeutic agent) which is sufficient to reduce and/or ameliorate the severity and/or duration of a condition ( e.g., a swine influenza 30 virus infection or a condition or symptom associated therewith, another infection e.g.,( another viral infection), an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition), prevent the advancement of a condition (e.g., a swine influenza virus infection or a condition or symptom associated therewith, another infection e.g.,( another viral infection), an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be used as a 35 vector to induce an immune response to a particular antigen associated with the condition), cause regression of a condition (e.g., a swine influenza virus infection or a condition or symptom associated therewith, another infection ( e.g., another viral infection) or an IFN treatable disease), prevent the recurrence, development, or onset of one or more symptoms associated with a condition e.g( ., a swine influenza virus infection or a condition or symptom associated therewith, another infection ( e.g., another viral infection) or an IFN treatable disease), reduce the titer of swine influenza 40 virus or enhance or improve the prophylactic or therapeutic effect(s) of another therapy ( e.g., prophylactic or therapeutic agent). [0050] As used herein, the term "epitopes" refers to sites or fragments of a polypeptide or protein having antigenic or immunogenic activity in an animal, preferably in a mammal, and most preferably in a pig. An epitope having immunogenic activity is a site or fragment of a polypeptide or protein that elicits an antibody response in an animal. An epitope having 45 antigenic activity is a site or fragment of a polypeptide or protein to which an antibody immunospecifically binds as determined by any method well-known to one of skill in the art, for example by immunoassays. [0051] As used herein, the term "fragment" in the context of a proteinaceous agent refers to a peptide or polypeptide comprising an amino acid sequence of at least 2 contiguous amino acid residues, at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous 50 amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid 55 residues of the amino acid sequence of a peptide, polypeptide or protein. In one embodiment, a fragment of a full-length protein retains activity of the full-length protein, e.g., IFN antagonist activity. In another embodiment, the fragment of the full-length protein does not retain the activity of the full-length protein, e.g., IFN antagonist activity. [0052] As used herein, the term "fragment" in the context of a nucleic acid refers to a nucleic acid comprising an nucleic

12 EP 2 497 492 B1

acid sequence of at least 2 contiguous nucleotides, at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, at least 15 contiguous nucleotides, at least 20 contiguous nucleotides, at least 25 contiguous nucleotides, at least 30 contiguous nucleotides, at least 35 contiguous nucleotides, at least 40 contiguous nucleotides, at least 50 contiguous nucleotides, at least 60 contiguous nucleotides, at least 70 contiguous nucleotides, at least contiguous 80 nucleotides, 5 at least 90 contiguous nucleotides, at least 100 contiguous nucleotides, at least 125 contiguous nucleotides, at least 150 contiguous nucleotides, at least 175 contiguous nucleotides, at least 200 contiguous nucleotides, at least 250 contiguous nucleotides, at least 300 contiguous nucleotides, at least 350 contiguous nucleotides, or at least 380 contiguous nucleotides of the nucleic acid sequence encoding a peptide, polypeptide or protein. In a preferred embodiment, a fragment of a nucleic acid encodes a peptide or polypeptide that retains activity of the full-length protein,e.g., IFN 10 antagonist activity. In another embodiment, the fragment of the full-length protein does not retain the activity of the full- length protein, e.g., IFN antagonist activity. [0053] As used herein, the phrase "heterologous sequence" refers to any sequence nucleic acid or protein, polypeptide or peptide sequence which is not normally found or in nature associated in nature with a nucleic acid or protein, polypeptide or peptide sequence of interest. For example, a "heterologous sequence" may refer to a sequence derived from a different 15 species. [0054] As used herein, the term "in combination" refers to the use of more than one therapye.g ( ., more than one prophylactic agent and/or therapeutic agent). The use of the term "in combination" does not restrict the order in which therapies (e.g., prophylactic and/or therapeutic agents) are administered to a subject with a conditione.g ( ., a swine influenza virus infection or a condition or symptom associated therewith, another infection ( e.g., another viral infection) 20 or an IFN treatable disease). A first therapy (e.g., a first prophylactic or therapeutic agent) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 we eks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks 25 after) the administration of a second therapy ( e.g., a second prophylactic or therapeutic agent) to a subject with a condition (e.g., a swine influenza virus infection or a condition or symptom associated therewith, another infection (e.g., another viral infection) or an IFN treatable disease). [0055] As used herein, the phrase "interferon antagonist activity" refers to a protein or polypeptide, or fragment, derivative, or analog thereof that reduces or inhibits the cellular interferon immune response. In particular, a protein or 30 polypeptide, or fragment, derivative, or analog thereof (e.g., swine influenza virus NS1) that has interferon antagonist activity reduces or inhibits interferon expression and/or activity. In a specific embodiment, the phrase "interferon antagonist activity" refers to a swine influenza virus protein or polypeptide, or fragment, derivative, or analog thereof that reduces or inhibits the cellular interferon immune response. A swine influenza viral protein or polypeptide with interferon antagonist activity may preferentially affect the expression and/or activity of one or two types of interferon (IFN). In one 35 embodiment, the expression and/or activity of IFN-α is affected. In another embodiment, the expression and/or activity of IFN-β is affected. In one embodiment, the expression and/or activity of IFN- γ is affected. In certain embodiments, the expression and/or activity of IFN- β and/or IFN- γ is reduced 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or more by protein, polypeptide, etc. with an interferon antagonist activity when compared to a control (e.g., PBS or a protein without interferon antagonist activity) in IFN-competent systems, e.g., a wild-type cell or animal 40 under the same conditions. In certain embodiments, the expression and/or activity of IFN-β and/or IFN-γ is reduced approximately 1 to approximately 100 fold, approximately 5 to approximately 80 fold, approximately 20 to approximately 80 fold, approximately 1 to approximately 10 fold, or approximately to approximately 5 fold, or approximately 40 to approximately 80 fold, or 1, 2, 3, 4, 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold by protein, polypeptide, etc. with an interferon antagonist activity when compared to a control e.g( ., PBS or a protein 45 without interferon antagonist activity) in IFN-competent systems under the same conditions. [0056] As used herein, the phrases "IFN-deficient systems" or "IFN-deficient substrates" refer to systems, e.g., cells, cell lines and animals, such as pigs, mice, chickens, turkeys, rabbits, rats, etc., which do not produce IFN or produce low levels of IFN (i.e., a reduction in IFN expression of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or more when compared to IFN-competent systems under the same conditions), do not respond or 50 respond less efficiently to IFN, and/or are deficient in the activity of one or more antiviral genes induced by IFN. [0057] As used herein, the phrase "IFN-inducing phenotype" refers to a phenotype whereby a virus demonstrates an increased cellular interferon response compared to a wild-type virus, which typically inhibits or reduces cellular interferon mediated responses. [0058] As used herein; the phrases "IFN treatable disorders", "IFN treatable diseases" and analogous phrases refer 55 to conditions that are preventable, treatable, managed or ameliorated by the administration of IFN, either IFN- α, β, γ or any combination thereof. The IFN treatable disorders need not be limited to swine, if swine influenza virus infects other species. Examples of IFN treatable disorders in pigs include, but are not limited to, foot and mouth disease, porcine Haemophilus pneumonia (PHP, and other disorders caused by infection with, e.g., infection by A. pleuropneumoniae,

13 EP 2 497 492 B1

P. haemolytica, P. multocida, H. somnus and A. suis. [0059] As used herein, the phrase "intermediate" phenotype with respect to IFN-antagonist activity refers to a phenotype which can stimulate a robust immune response, while being attenuated because viruses with an intermediate phenotype cannot overcome the host IFN response. In particular, an intermediate phenotype can stimulate an immune response 5 and inhibit/reduce IFN only to the extent that it allows fewer rounds of virus replication or lower numbers of virus particles being produced compared to wild-type virus, as determined by techniques well-known in the art ( e.g., as measured by lower plaque titer or hemagglutination assays). [0060] As used herein, the term "isolated", in the context of viruses, refers to a virus that is derived from a single parental virus. A virus can be isolated using routine methods known to one of skill in the art including, but not limited to, 10 those based on plaque purification and limiting dilution. [0061] As used herein, the terms "manage," "managing," and "management" refer to the beneficial effects that a subject derives from a therapy ( e.g., a prophylactic or therapeutic agent), which does not result in a cure of the disease. In certain embodiments, a subject is administered one or more therapies ( e.g., one or more prophylactic or therapeutic agents) to "manage" a disease so as to prevent the progression or worsening of the disease. 15 [0062] As used herein, the phrase "multiplicity of infection" or "MOI" is the average number of virus per infected cell. The MOI is determined by dividing the number of virus added (ml added x PFU) by the number of cells added (ml added x cells/ml). [0063] As used herein, the phrase "non-swine influenza virus" in the context of swine influenza viruses containing or comprising non-swine influenza virus sequences refers to any influenza virus strain or isolate comprising a sequence 20 (e.g., a nucleic acid or amino acid sequence) heterologous to swine influenza virus,i.e, the sequence is not found to naturally in association with swine influenza virus. [0064] As used herein, the phrase "NS1 gene" refers to the gene which encodes the nonstructural protein (NS 1) in influenza. NS1 is one of the eight molecules encoded by the segmented genome of influenza A and other viruses. A "swine influenza virus NS1 gene," is an NS1 gene isolated from a swine influenza virus. Representative swine NS1 25 genes can be found in public sequence databases such as Genbank and include, but are not limited to, Genbank Accession No. AJ293939 (A/swine/Italy/13962/95(H3N2)) and Genbank Accession No. AJ344041 (A/swine/Cotes d’Ar- mor/1121/00(H1N1)). A "NS1 gene product" refers to a gene product ( e.g., a RNA or protein) encoded by a NS1 gene. In the case of a protein, the NS1 gene product is full-length and has wild-type NS1 activity,e.g., ( from Influenza A/swine/Texas/4199-2/98. A "swine influenza virus NS1 gene product" refers to a gene product ( e.g., a RNA or protein) 30 encoded by a swine influenza virus NS1 gene. In the case of a protein, the swine influenza virus NS 1 gene product is full-length and has wild-type swine influenza virus NS1 activity, e.g., from Influenza A/swine/Texas/4199-2/98. [0065] As used herein, the terms "prevent", "preventing," and "prevention" refer to the inhibition of the development or onset of a condition (e.g, a swine influenza virus infection or a condition associated therewith, an infection other than a swine influenza virus infection or a condition associated therewith, an IFN-treatable disease or a condition in which 35 the attenuated swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition), or the prevention of the recurrence, onset, or development of one or more symptoms of a condition (e.g., a swine influenza virus infection or a condition associated therewith, an infection other than a swine influenza virus infection or a condition associated therewith, or an IFN-treatable disease), in a subject resulting from the administration of a therapy ( e.g., a prophylactic or therapeutic agent), or the administration of a combination of therapies 40 (e.g., a combination of prophylactic or therapeutic agents). [0066] As used herein, the terms "prophylactic agent" and "prophylactic agents" refer to any agent(s) which can be used in the prevention of a condition or a symptom thereofe.g., ( a swine influenza virus infection or a condition or symptom associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be 45 used as a vector to induce an immune response to a particular antigen associated with the condition). Preferably, a prophylactic agent is an agent which is known to be useful to or has been or is currently being used to the prevent or impede the onset, development, progression and/or severity of a swine influenza virus infection or a condition or symptom associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be used as a 50 vector to induce an immune response to a particular antigen associated with the condition. [0067] As used herein, the term "prophylactically effective amount" refers to the amount of a therapy ( e.g., prophylactic agent) which is sufficient to result in the prevention of the development, recurrence, or onset of a condition or a symptom thereof (e.g., a swine influenza virus infection or a condition or symptom associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition 55 in which the attenuated swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition) or to enhance or improve the prophylactic effect(s) of another therapye.g ( ., a prophylactic agent). [0068] As used herein, the phrase "purified" in the context of viruses refers to a virus which is substantially free of

14 EP 2 497 492 B1

cellular material and culture media from the cell or tissue source from which the virus is derived. The language "substantially free of cellular material" includes preparations of virus in which the virus is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, virus that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of cellular protein (also 5 referred to herein as a "contaminating protein"). The virus is also substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the virus preparation. A virus can be purified using routine methods known to one of skill in the art including, but not limited to, chromatography and centrifugation. [0069] As used herein, the terms "subject" or "patient" are used interchangeably. As used herein, the terms "subject" and "subjects" refers to an animal (e.g., birds, reptiles, and mammals), preferably a mammal including a non-primate 10 (e.g., a camel, donkey, zebra, cow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human). The subject or patient may have a swine influenza virus infection. The mammal may be 0 to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to 10 years old, 10 to 15 years old, 15 to 20 years old, 20 to 25 years old, 25 to 30 years old, 30 to 35 years old, 35 to 40 years old, 40 to 45 years old, 45 to 50 years old, 50 to 55 years old, 55 to 60 years old, 60 to 65 years old, 65 to 70 years old, 70 to 75 years old, 75 to 80 years old, 80 to 85 years old, 85 to 90 15 years old, 90 to 95 years old or 95 to 100. The subject or patient may be a pig. The pig may be 0 to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to 10 years old or 10 to 15 years old. The natural lifespan of a pig is 10-15 years. [0070] As used herein, "swine influenza virus" refers to a type A or type C influenza virus from the family orthomyxovirus that causes swine influenza. While orthomyxovirus has three groups: type A, type B and type C, only type A and type C influenza viruses infect pigs. Subtypes of swine influenza virus include H1N1, H1N2, H3N2, and H3N1. H9N2 and 20 H5N1 can also be found in pigs. A swine influenza virus may be an influenza virus that has been isolated from swine. A swine influenza virus may contain a swine NS1 gene. Repres entative swine NS1 genes can be found in public sequence databases such as Genbank and include, but are not limited to, Genbank Accession No. AJ293939 (A/swine/Ita- ly/13962/95(H3N2)) and Genbank Accession No. AJ344041 (A/swine/Cotes d’Armor/1121/00(H1N1)). Examples of swine influenza virus variants include, but are not limited to, A/Swine/Colorado/1/77, A/Swine/Colorado/23619/99, 25 A/Swine/Cote d’Armor/3633/84, A/Swine/England/195852/92, A/Swine/Finistere/2899/82, A/Swine/Hong Kong/10/98, A/Swine/Hong Kong/9/98, A/Swine/Hong Kong/81/78, A/Swine/Illinois/100084/01, A/Swine/Illinois/100085A/01, A/Swine/Illinois/21587/99, A/Swine/Indiana/1.726/88, A/Swine/Indiaria/9K035199, A/Swine/Indiana/P12439/00, A/Swine/Iowa/30, A/Swine/lowa/15/30, A/Swine/lowa/533/99, A/Swine/Iowa/569/99, A/Swine/lowa/3421/90, A/Swine/lowa/8548-1/98, A/Swine/lowa/930/01, A/Swine/lowa/17672/88, A/Swine/Italy/1513-1/98, A/Swine/Ita- 30 ly/1523/98, A/Swine/Korea/CY02/02, A/Swine/Minnesota/55551/00, A/Swine/Minnesota/593/99, A/Swine/Minneso- ta/9088-2/98, A/Swine/Nebraska/1/92, A/Swine/Nebraska/209/98, A/Swine/Netherlands/12/85, A/Swine/North Caroli- na/16497/99, A/Swine/North Carolina/35922/98, A/Swine/North Carolina/93523/01, A/Swine/North Carolina/98225/01, A/Swine/Oedenrode/7C/96, A/Swine/Ohio/891/01, A/Swine/Oklahoma/18717/99, A/Swine/Oklahoma/18089/99, A/Swine/Ontario/01911-1/99, A/Swine/Ontario/01911-2/99, A/Swine/Ontario/41848/97, A/Swine/Ontario/97, 35 A/Swine/Quebec/192/81, A/Swine/Quebec/192/91, A/Swine/Quebec/5393/91, A/Swine/Taiwan/7310/70, A/Swine/Ten- nessee/24/77, A/Swine/Texas/4199-2/98, A/Swine/Wisconsin/125/97, A/Swine/Wisconsin/136/97, A/Swine/Wiscon- sin/163/97, A/Swine/Wisconsin/164/97, A/Swine/Wisconsin/166/97, A/Swine/Wisconsin/168/97, A/Swine/Wiscon- sin/235/97, A/Swine/Wisconsin/238/97, A/Swine/Wisconsin/457/98, A/Swine/Wisconsin/458/98, A/Swine/Wiscon- sin/464/98 and A/Swine/Wisconsin/14094/99. 40 [0071] As used herein, the term "synergistic" refers to a combination of therapies (e.g., prophylactic or therapeutic agents) which is more effective than the additive effects of any two or more single therapies (e.g., one or more prophylactic ortherapeutic agents). A synergistic effect of a combination of therapies ( e.g., a combination of prophylactic or therapeutic agents) permits the use of lower dosages of one or more of therapies (e.g., one or more prophylactic or therapeutic agents) and/or less frequent administration of said therapies to a subject with a condition ( e.g., a swine influenza virus 45 infection or a condition or symptom associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition). The ability to utilize lower dosages of therapies ( e.g., prophylactic or therapeutic agents) and/or to administer said therapies less frequently reduces the toxicity associated with the administration of said therapies to a subject without 50 reducing the efficacy of said therapies in the prevention or treatment of a condition ( e.g., a swine influenza virus infection or a condition or symptom associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition). In addition, a synergistic effect can result in improved efficacy of therapies ( e.g., prophylactic or therapeutic agents) in the prevention 55 or treatment of a condition (e.g., a swine influenza virus infection or a condition or symptoms associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition). Finally, synergistic effect of a combination of therapies

15 EP 2 497 492 B1

(e.g., prophylactic or therapeutic agents) may avoid or reduce adverse or unwanted side effects associated with the use of any single therapy. [0072] As used herein, the term "T cell receptor modulator" refers to an agent which modulates the phosphorylation of a T cell receptor, the activation of a signal transduction pathway associated with a T cell receptor and/or the expression 5 of a particular protein such as a cytokine. Such an agent may directly or indirectly modulate the phosphorylation of a T cell receptor, the activation of a signal transduction pathway associated with a T cell receptor, and/or the expression of a particular protein such as a cytokine. Examples of T cell receptor modulators include, but are not limited to, peptides, polypeptides, proteins, fusion proteins and antibodies which immunospecifically bind to a T cell receptor or a fragment thereof. Further, examples of T cell receptor modulators include, but are not limited to, proteins, peptides, polypeptides 10 (e.g., soluble T cell receptors), fusion proteins and antibodies that immunospecifically bind to a ligand for a T cell receptor or a fragment thereof. [0073] As used herein, the term "therapeutically effective amount" refers to the amount of a therapy, which is sufficient to reduce the severity of a condition e.g( ., a swine influenza virus infection or a condition associated therewith e.g( ., influenza induced abortions, depression, inappetance, anorexia, dyspnea, myalgia, or enlarged submandibular lymph 15 nodes), an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition), reduce the duration of a condition ( e.g., swine influenza virus infection or a condition associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition in which the attenuated swine 20 influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition), reduce the titer of swine influenza virus or other virus, reduce the number of other pathogens, ameliorate one or more symptoms of a condition e.g.,( a swine influenza virus infection or a condition associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be used as a vector to induce an immune 25 response to a particular antigen associated with the condition), prevent the advancement of a condition (e.g., a swine influenza virus infection or a condition associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition), cause regression of a condition (e.g., a swine influenza virus infection or a condition associated therewith, 30 an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition), or enhance or improve the therapeutic effect(s) of another therapy. [0074] As used herein, the terms "therapies" and "therapy" can refer to any protocol(s), method(s), compositions, 35 formulations, and/or agent(s) that can be used in the prevention, treatment, management, or amelioration of a condition (e.g., a swine influenza virus infection or a condition or symptom associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition). In certain embodiments, the terms "therapies" and "therapy" refer to biological therapy, 40 supportive therapy, and/or other therapies useful in treatment, management, prevention, or amelioration of a swine influenza virus infection or a condition or symptom associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition, known to one of skill in the art. 45 [0075] As used herein, the terms "therapeutic agent" and "therapeutic agents" refer to any agent(s) which can be used in the prevention, treatment, management, or amelioration of a condition or a symptom thereof ( e.g., a swine influenza infection or a condition or symptoms associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the 50 condition). Preferably, a therapeutic agent is an agent which is known to be useful for, or has been or is currently being used for the prevention, treatment, management, or amelioration of a swine influenza virus infection or a condition or symptoms associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition. 55 [0076] As used herein, the terms "treat," "treatment," and "treating" refer to the eradication or control of swine influenza virus replication or the replication of a pathogen ( e.g., a virus) other than swine influenza virus, the reduction in the titer of swine influenza virus or virus other than swine influenza virus, the reduction in the numbers of a pathogen, the reduction or amelioration of the progression, severity, and/or duration of a condition e.g.,( a swine influenza virus infection or a

16 EP 2 497 492 B1

condition associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease or a condition in which the attenuated swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition), or the amelioration of one or more symptoms resulting from the administration of one or more therapies (including, but not limited to, the 5 administration of one or more prophylactic or therapeutic agents). [0077] The term "tumor antigen" as used herein refers to a molecule on a tumor cell that can be specifically recognized by immune T cells or antibodies. A tumor antigen includes those present only on tumor cells (tumor specific antigens) as well as those present on normal cells but expressed preferentially or aberrantly on tumor cells (tumor associated antigens). Examples of tumor antigens include, but are not limited to, antigens of sarcoids, prostate cancer, fibrosarcoma, 10 self-differentiation antigens such as oncofetal, or differentiation, antigens which are expressed by malignant cells, including but not limited to oncofetal antigens such as carcinoembryonio antigens (CEA) of the colon, alpha-fetoprotein, the human antigenic counterpart or functional equivalent of the 175 kDa murine antigen of transitional cell bladder carcinomas, the melanoma associated antigen p97 or GD3, and differentiation antigens of human lung carcinomas such as L6 and L20. 15 [0078] As used herein, the phrase "wild-type swine influenza virus" refers to the types of an swine virus that are prevalent, circulating naturally and producing typical outbreaks of disease. Examples of wild-type swine influenza viruses include, but are not limited to, A/Swine/Colorado/1/77, A/Swine/Colorado/23619/99, A/Swine/Cote d’Armor/3633/84, A/Swine/England/195852/92, A/Swine/Finistere/2899/82, A/Swine/Hong Kong/10/98, A/Swine/Hong Kong/9/98, A/Swine/Hong Kong/81/78, A/Swine/Illinois/100084/01, A/Swine/Illinois/100085A/01, A/Swine/Illinois/21587/99, 20 A/Swine/Indiana/1726/88, A/Swine/Indiana/9K035/99, A/Swine/Indiana/P12439/00, A/Swine/Iowa/30, A/Swine/Io- wa/15/30, A/Swine/Iowa/533/99, A/Swine/Iowa/569/99, A/Swine/Iowa/3421/90, A/Swine/Iowa/8548-1/98, A/Swine/Io- wa/930/01, AlSwine/Iowa/17672/88, A/Swine/Italy/1513-1/98, A/Swine/Italy/1523/98, A/Swine/Korea/CY02/02, A/Swine/Minnesota/55551/00, A/Swine/Minnesota/593/99, A/Swine/Minnesota/9088-2/98, A/Swine/Nebraska/1/92, A/Swine/Nebraska/209/98, A/Swine/Netherlands/12/85, A/Swine/North Carolina/16497/99, A/Swine/North Caroli- 25 na/35922/98, A/Swine/North Carolina/93523/01, A/Swine/North Carolina/98225/01, A/Swine/Oedenrode/7C/96, A/Swine/Ohio/891/01, A/Swine/Oklahoma/18717/99; A/Swine/Oklahoma/18089/99, A/Swine/Ontario/01911-1/99, A/Swine/Ontario/01911-2/99, A/Swine/Ontario/41848/97, A/Swine/Ontario/97, A/Swine/Quebec/192/81, A/Swine/Que- bec/192/91, A/Swine/Quebec/5393/91, A/Swine/Taiwan/7310/70, A/Swine/Tennessee/24/77, A/Swine/Tex- as/4199-2/98, A/Swine/Wisconsin/125/97, A/Swine/Wisconsin/136/97, A/Swine/Wisconsin/163/97, A/Swine/Wiscon- 30 sin/164/97, A/Swine/Wisconsin/166/97, A/Swine/Wisconsin/168/97, A/Swine/Wisconsin/235/97, A/Swine/Wiscon- sin/238/97, A/Swine/Wisconsin/457/98, A/Swine/Wisconsin/458/98, A/Swine/Wisconsin/464/98 and A/Swine/Wiscon- sin/14094/99.

4. DESCRIPTION OF THE FIGURES 35 [0079]

Figures 1A-1B. Generation of plasmid-derived Sw/Tx/98 influenza viruses with NS1 mutated proteins. Figure 1A depicts a schematic diagram of the wild type and mutated NS influenza gene segments. The NS gene segment 40 was modified to create mutated NS1 genes encoding 73, 99 and 126 aa, respectively. NS1 mutations did riot affect the sequence of the NEP protein. Underlined sequences were introduced to generate stop codons (in bold). Figure 1B depicts RT-PCR analysis of the rWT and NS1 deletion mutant viruses. Influenza viral RNA segments were amplified using primers specific for the non-coding regions of all 8 influenza viral segments. Arrows indicate the decreasing size of the NS segment. Dots indicate a product corresponding to the 3’ end of the HA gene segment 45 due to internal binding of the forward primer. Figures 2A-2D. Characterization of plasmid-derived wild-type and NS1 mutant Sw/TX/98 viruses in tissue culture. Figure 2A shows multi-cycle growth curves of wild-type and NS1 deletion mutants in PK-15 cells. Figure 2B shows single cycle growth curves of wild-type and NS1 deletion mutants in PK-15 cells. Figure 2C shows plaque size in MDCK cells at 3 days post infection. Figure 2D shows a time course of viral protein expression. PK-15 cells 50 infected with rWT and NS1 mutant viruses (MOI=2) and labeled with 35[ S]Met-Cys at the indicated times p.i. Cell extracts were subjected to SDS-PAGE and analyzed by autoradiography. Figures 3A-3C. Induction of type I IFN In PK-15 cells infected with plasmid-derived wild-type and NS1 mutant Sw/TX/98 viruses. Figure 3A shows a schematic representation of the IFN-bioassay levels of IFN secreted by virus infected cells in a fluorescent assay. Figure 3B shows a time course of type I IFN synthesis. Fresh PK-15 cells were 55 treated for 24 hours with UV-inactivated supernatants (harvested at different times p.i.) from PK-15 cells which were infected with the indicated viruses, followed by VSV-GFP infection. Sixteen hours post infection, cells expressing GFP were visualized by fluorescence microscopy. the induction of IFN and TNF-α. Figure 3C shows a RT-PCR analysis of TNF-α, IFN-β- and β-actin-specific mRNAs in virus-infected PK-15 cells.

17 EP 2 497 492 B1

Figures 4A-4C. Infection of 4-week-old pigs with plasmid-derived wild-type and NS1 mutant Sw/TX/98 viruses. Figure 4A shows a bar chart depicting the histopathological scores of lungs from pigs infected with plasmid- derived TX/98 deletion viruses. Mean percentage (6SEM) of lung surface with microscopic lesions on day 5 p.i. Figure 4B shows microscopic lesions in the medium bronchioles on day 5 p.i. Figure 4C shows virus titers in 5 bronchoalveolar lavage fluid on day 5 p.i. Figures 5A-5D. Histopathological examination of lungs from pigs infected with plasmid-derived wild-type and NS1 mutant Sw/TX/98 viruses, magnification 200x. Figure 5A shows medium-sized bronchiole from the lung of a non-infected control pig. Epithelial lining is intact, and surrounding alveoli are normal. Figure 5B shows severe acute necrotizing bronchiolitis and interstitial pneumonia characteristic of the widespread lesion induced by infection 10 with rWT TX/98 virus. Figure 5C shows one normal bronchiole and one affected bronchiole in early recovery from infection with deletion mutant 1-99 virus, representative of the less extensive injury induced by infection with this virus. Figure 5D shows normal bronchiole and surrounding alveoli from the lung of a pig inoculated with 1-126 virus. No lesions were induced by this virus. Figures 6A-D. Histopathological examination of lungs from pigs infected with plasmid-derived wild-type 15 and NS1 mutant Sw/TX/98 viruses, magnification 200x. Figure 6A shows the epithelial lining of a larger bronchiole from the lung of a non-infected control pig. Epithelial cells are tall columnar with pseudo-stratified basal nuclei and prominent surface cilia. Figure 6B shows the epithelial lining from a larger bronchiole from the lung of a pig infected with TX/98 rWT virus. Epithelial lining is in total disarray due to necrosis and regenerative proliferation of the epithelial cells. Figure 6C shows a large bronchiole and smaller branching airway from the lung of a pig infected with deletion 20 mutant 1-99 virus, representative of the less extensive injury induced by this virus. Figure 6D shows a normal epithelial lining from the lung of a pig inoculated with 1-126 virus. No bronchiolar injury resulted. Figure 7. Histopathology of TX/98/del 126-vaccinated pigs. Figure 7 shows a bar chart depicting the histopathological scores of lungs from pigs vaccinated with TX/98/del126. Mock = non vaccinated, sham-inoculated pigs; H3N2 = non vaccinated pigs inoculated with 2x10 5 PFU per pig with A/Swine/Texas/4199-2/98; H1N1= non-vaccinated pigs 25 inoculated with 2x105 PFU per pig with A/Swine/MN/37866/99; MLV + Mock = TX/98/del 126 vaccinated, sham inoculated pigs; MLV + H3N2 = TX/98/del 126 vaccinated pigs inoculated with 2x10 5 PFU per pig with A/Swine/Tex- as/4199-2/98; MLV+ H1N1= TX/98/del 126 vaccinated pigs inoculated with5 PFU 2x10 per pig with A/Swine/MN/37866/99. Figure 8: Immunohistochemistry for SIV-antigen in TX/98/del 126-vaccinated pigs. Figure 8 shows a bar chart 30 depicting the immunohistological scores for pigs vaccinated with TX/98/del126. Mock = non vaccinated, sham- inoculated pigs; H3N2 = non vaccinated pigs inoculated with 2x105 PFU per pig with A/Swine/Texas/4199-2/98; H1N1= non-vaccinated pigs inoculated with 2x10 5 PFU per pig with A/Swine/MN/37866/99; MLV + Mock = TX/98/del 126 vaccinated, sham inoculated pigs; MLV + H3N2 = TX/98/del 126 vaccinated pigs inoculated with 2x105 PFU per pig with A/Swine/Texas/4199-2/98; MLV+ H1N1= TX/98/del 126 vaccinated pigs inoculated with 2x10 5 PFU per 35 pig with A/Swine/MN/37866/99. Figure 9: Virus titers in long lavage of TX/98/del 126-immunized pigs challenged with H3N2 and H1N1 SIVs. Figure 9 shows a bar chart depicting the viral titers in lung lavage from pigs vaccinated with TX/98/del126. Mock = non vaccinated, sham-inoculated pigs; H3N2 = non vaccinated pigs inoculated with 2x105 PFU per pig with A/Swine/Tex-as/4199-2/98; H1N1= non-vaccinated pigs inoculated with5 PFU 2x10 per pig with 40 A/Swine/MN/37866/99; MLV + Mock = TX/98/del 126 vaccinated, sham inoculated pigs; MLV + H3N2 = TX/98/del 126 vaccinated pigs inoculated with 2x10 5 PFU per pig with A/Swine/Texas/4199-2/98; MLV+H1N1= TX/98/del 126 vaccinated pigs inoculated with 2x10 5 PFU per pig with A/Swine/MN/37866/99. The limit of virus detection was 10 1.5

TCID50/ml.

45 5. DETAILED DESCRIPTION OF THE INVENTION

[0080] The present invention provides a genetically engineered attenuated swine influenza virus having an altered interferon antagonist phenotype, wherein said virus comprises a swine influenza virus A/Swine/Texas/4199-2/98 NS1 gene with a mutation resulting in an NS1 protein as set out in claim 1. 50 [0081] The present invention further provides an immunogenic formulation comprising the genetically engineered attenuated swine influenza virus, and a physiologically acceptable excipient. Also, the present invention provides a pharmaceutical formulation comprising the genetically engineered attenuated swine influenza virus of the present invention and a pharmaceutically acceptable carrier. [0082] Furthermore, the present invention provides use of an effective amount of the immunogenic formulation of the 55 present invention for use in immunizing or inducing an immune response in a pig. Also, the present invention provides an effective amount of the genetically engineered attenuated swine influenza virus of the present invention (a) for use in the prevention of a condition or a symptom associated with swine influenza virus in a pig; or (b) for use in the treatment of a swine influenza virus infection in a pig.

18 EP 2 497 492 B1

[0083] The present invention also provides a method for production of an immunogenic formulation comprising (a) propagating in a substrate the genetically engineered attenuated swine influenza virus of the present invention and (b) collecting progeny virus, wherein the substrate is a cell, cell line or embryonated egg. [0084] Furthermore, the present invention provides a cultured cell containing the genetically engineered attenuated 5 swine influenza virus of the present invention. The present invention also provides an embryonated egg containing the genetically engineered attenuated swine influenza virus of the present invention. Disclosed herein are attenuated swine influenza viruses having an impaired ability to antagonize the cellular interferon (IFN) response, methods for producing such attenuated swine influenza viruses, and the use of such viruses in vaccine and pharmaceutical formulations. Such viruses are capable of generating an immune response and creating immunity but not causing illness, or causing fewer 10 and/or less severe symptoms, i.e., the viruses have decreased virulence. Therefore, they are ideal candidates for live virus vaccines. Moreover, the attenuated swine influenza viruses can induce a robust IFN response which has other biological consequences in vivo, affording protection against subsequent infectious diseases and/or inducing antitumor responses. Therefore, the attenuated swine influenza viruses can be used pharmaceutically, for the prevention or treatment of other infectious diseases and/or IFN-treatable diseases. 15 [0085] The invention is based, in part, on the Applicants’ discovery that swine influenza viruses engineered to contain or containing a deletion(s) in the NS1 gene have impaired replication relative to wild-type swine influenza viruses as demonstrated by fewer lung lesions upon infection of pigs, reduced viral titers in bronchoalveolar lavage fluid (BALF) and reduced detection of virus on nasal swabs. Surprisingly, and contrary to results seen with human influenza virus in mouse models, the length of the NS1 protein does not correlate with the level of attenuation of the swine influenza virus. 20 Applicants have discovered that with swine influenza, a mutant virus with the shortest NS1 protein is the least attenuated. In other words, swine influenza viruses containing shorter deletions in the NS1 gene exhibit a greater attenuation in vivo than swine influenza viruses containing longerdeletions in their NS1 gene, orwild-type swine influenza viruses. Applicants have further discovered that the recombinant swine influenza viruses are impaired in their ability to inhibit IFN production in vitro, and they do do not replicate as efficiently as the parental recombinant strain in 10-day old embryonated hen 25 eggs, in MDCK cells, in PK-15 cells or in an in vivo pig model. While not intending to be bound to any theory or explanation for the mechanism of action of the swine influenza virus NS1 deletion mutants in vivo, the attenuated features of such viruses are presumably due to their levels of NS1 protein expression, their ability to induce a robust cellular IFN response, and their impaired ability to antagonize such a response. However, the beneficial features of such viruses may not be solely attributable to their effect on the cellular interferon response. Indeed, alterations in other activities associated wit h 30 NS1, such as, alteration of pre-mRNA splicing, inhibition of cellular mRNA polyadenylation, poly(A)-containing mRNA nucleocytoplasmic transport, and stimulation of viral protein synthesis, may contribute to the desired attenuated phenotype achieved by the introduction of mutations in the NS1 gene of swine influenza virus. [0086] An attenuated swine influenza disclosed herein comprises a mutation in a swine influenza NS1 gene that diminishes the ability of the NS1 gene product to antagonize the cellular interferon response. In one aspect, an attenuated 35 swine influenza virus disclosed herein comprises a genome comprising a mutation in a swine influenza virus NS1 gene that diminishes the ability of the NS 1 gene product to antagonize a cellular interferon response, and permits the attenuated virus, at a multiplicity of infection (MOI) of between 0.0005 and 0.001, 0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or a MOI of 0.0005, 0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately 1 to approximately 100 fold, approximately 5 to approximately 80 fold, approximately 20 40 to approximately 80 fold, or approximately 40 to approximately 80 fold, approximately 1 to approximately 10 fold, approximately 1 to approximately 5 fold, approximately 1 to approximately 4 fold, approximately to approximately 3 fold, approximately 1 to approximately 2 fold, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold lower than wild-type swine influenza virus in cells (e.g., cells of a humane.g ( ., PerC6, a producer cell line derived from human embryonic retinoblasts transformed with the E1 region of Adenovirus 45 5), mouse, chicken ( e.g., chicken embryo fibroblasts), rat, birds, or pig (e.g., PK(D1) cells, PK(15) cells, PK13 cells, NSK cells, LLC-PK1 cells, LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells, PK-2a/CL 13 or SJPL cells)), as determined approximately 2 to 10 days, 3 to 7 days, 3 to 5 days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 days post-infection when propagated under the same conditions. The titers of attenuated and wild-type swine influenza viruses can be determined utilizing any technique well-known in the art or described herein, (e.g., hemagglutination assays, plaque assays, egg infectious 50 doses (EID50), tissue culture infectious doses (TCID50), etc.) and the viruses can be propagated under conditions described herein or well-known in the art (e.g., in pig cells, MDCK cells (e.g., in MEM, 10% v/v fetal calf serum (FCS), 1% penicillin/ streptomycin at 37°C in a 5% CO 2 humidified incubator) or embryonated chicken eggs ( e.g., in a stationary incubator at 37°C with 55% relative humidity). Alternatively, the viruses can be propagated in cells e.g( ., in pig cells, MDCK cells, etc.) that are grown in serum-free or serum reducede.g (., TPCK trypsin) medium. The growth of an 55 attenuated swine influenza virus of the invention may be compared to a particular standard or reference, e.g., wild-type swine influenza virus A/Swine/Texas/4199-2/98. [0087] In a specific aspect, an attenuated swine influenza virus disclosed herein comprises a genome comprising a mutation in a swine influenza virus NS1 gene that diminishes the ab ility of the NS1 g ene product to antagonize a cellular

19 EP 2 497 492 B1

interferon response, and permits the attenuated virus, at a multiplicity of infection (MOI) of between 0.0005 and 0.001, 0.001 and 0.01,0.01 and 0.1, or 0.1 and 1, or a MOI of 0.0005, 0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,4.0, 4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately 1 to approximately 100 fold, approximately 5 to approximately 80 fold, approximately 20 to approximately 80 fold, or approximately 40 to approximately 80 fold, 5 approximately 1 to approximately 10 fold, Approximately 1 to approximately 5 fold, approximately 1 to approximately 4 fold, approximately 1 to approximately 3 fold, approximately 1 to approximately 2 fold, or approximately 1, 2, 3, 4, 5, 6, 7, 8,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold lower than wild-type swine influenza virus in pig cells, as determined approximately 2 to 10 days, 3 to 7 days, 3 to 5 days, or 2, 3,4, 5, 6, 7, 8, 9, 10 days post-infection when propagated under the same conditions. The titers of attenuated and wild-type swine influenza viruses 10 can be determined utilizing any technique well-known in the art or described herein,e.g ( ., hemagglutination assays, plaque assays, egg infectious doses (EID50), tissue culture infectious doses (TCID50), etc.) and the viruses can be propagated under conditions described herein or well-known in the art (e.g., in pig cells, MDCK cells ( e.g., in MEM, 10%

v/v fetal calf serum (FCS), 1% penicillin/streptomycin at 37°C in a 5% CO 2 humidified incubator) or embryonated chicken eggs (e.g., in a stationary incubator at 37°C with 55% relative humidity). Alternatively, the viruses can be propagated in 15 cells (e.g., in pig cells, MDCK cells, etc.) that are grown in serum-free or serum reduced ( e.g., TPCK trypsin) medium. [0088] An attenuated swine influenza virus having the desired phenotype can itself be used as the active ingredient in vaccine, pharmaceutical or immunogenic formulations. Alternatively, the attenuated swine influenza virus can be used as the vector or "backbone" of recombinantly produced vaccines or immunogenic formulations. To this end, the "reverse genetics" technique can be used to engineer mutations or introduce foreign epitopes into the attenuated swine influenza 20 virus, which would serve as the "parental" strain. In this way, vaccines can be designed for immunization against strain variants, or in the alternative, against completely different infectious agents or disease antigens ( e.g., tumor antigens). For example, the attenuated virus can be engineered to express neutralizing epitopes of other preselected strains. Alternatively, epitopes of other viruses can be built into the attenuated swine influenza virus. Alternatively, epitopes of non-viral infectious pathogens (e.g., parasites, bacteria, fungi) can be engineered into the attenuated swine influenza 25 virus. [0089] Disclosed herein are methods for vaccinating a subject comprising administering the vaccine formulations disclosed herein. In one aspect, disclosed herein is a method comprising administering to a subject an effective amount of a vaccine formulation disclosed herein. In certain aspects, the dose of the vaccine formulation administered to the subject is between about 102 to about 108, about 103 to about 107, or about 104 to about 5 x 106 pfu. In other aspects, 30 the dose of the vaccine formulation administered is 102, 5 x 102, 103, 5 x 103, 104, 5 x 104, 105, 5 x 105, 106, 5 x 106, 107, 5 x 107 or 108 pfu. In other aspects, the dose of an immunogenic formulation of the invention administered to a subject is 102 to about 108, about 103 to about 107, or about 104 to about 5 x 106 pfu. In yet other aspects, the dose of an immunogenic formulation of the invention administered to a subject is 10 2, 5 x 102, 103, 5 x 103, 104, 5 x 104, 105, 5 x 105, 106, 5 x 106, 107, 5 x 107 or 108 pfu. In specific aspects, the subject is a donkey, zebra, camel, dog, avian (e.g., 35 a duck). In a preferred aspects, the subjects is a pig. [0090] The present invention provides immunogenic formulations comprising an attenuated swine influenza virus of the invention Disclosed herein are also methods for inducing an immune response for the treatment, management or prevention of a swine influenza virus infection or a condition or symptom associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, or a condition in which the attenuated 40 swine influenza viruses can be used as a vector to induce an immune response to a particular antigen associated with the condition, comprising administering to a subject an immunogenic formulation of the invention. The present invention relates to a immunogenic formulation for use in a method for immunizing or inducing an immune response e.g., for the treatment, management or prevention of a swine influenza virus infection or a condition or symptom associated therewith, comprising administering to a subject an effective amount of an immunogenic formulation of the invention. The dose of 45 immunogenic formulation to be administered to the subject may be between about 10 2 to about 10 8, about 103 to about 107, about 104 to about 5 x 10 6 pfu or about 10 4 to about 10 7 pfu. The dose of immunogenic formulation of the invention to be adminitered [0091] to a subjec may be 102, 5 x 102, 103, 5 x 103, 104, 5 x 104, 105, 5 x 105, 106, 5 x 106, 107, 5 x 107 or 108 pfu. The subject may be a donkey, zebra, camel, dog, avian (e.g., a duck), and especially a pig. 50 [0092] The attenuated swine influenza viruses, which induce robust IFN responses in subjects, may also be used in pharmaceutical formulations for the prophylaxis or treatment of other infections, or IFN-treatable diseases such as cancer. In this regard, the tropism of the attenuated swine influenza virus can be altered to target the virus to a desired target organ, tissue or cells in vivo or ex vivo. Using this approach, the IFN response can be induced locally, at the target site, thus avoiding or minimizing the side effects of systemic IFN treatments. To this end, the attenuated swine influenza 55 virus can be engineered to express a ligand specific for a receptor of the target organ, tissue or cells. [0093] Disclosed herein are methods for preventing, managing or treating an infection or IFN-treatable disease in a subject, other than a swine influenza viral infection or IFN-treatable disease caused by swine influenza virus, comprising administering a pharmaceutical formulation of the invention. In one aspect, disclosed herein is a method for preventing,

20 EP 2 497 492 B1

managing or treating an infection or IFN-treatable disease in a subject, other than a swine influenza viral infection or IFN-treatable disease caused by swine influenza virus, comprising administering an effective amount of a pharmaceutical formulation of the invention. The dose of the pharmaceutical formulation to be administered to the subject may be between about 102 to about 108, about 103 to about 107, about 104 to about 5 x 106 pfu or about 104 to about 1012 pfu. 5 The dose of the pharmaceutical formulation to be administered to the subject may be 5 x 10 2, 103, 5 x 10 3, 104, 5 x 10 4, 105, 5 x 105, 106, 5 x 106, 107, 5 x 107, 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011, 5 x 1011 or 1012 pfu. The subject may be a donkey, zebra, camel, dog, avian (e.g., a duck), and especially a pig. [0094] Disclosed herein are methods for preventing, managing or treating cancer in a subject comprising administering a pharmaceutical composition of the invention. In one aspect, disclosed herein is a method for preventing, managing or 10 treatingcancer in a subject comprising administering an effective amount of a pharmaceuticalcomposition of the invention. The dose of the pharmaceutical formulation to be administered to the subject may be between about 10 2 to about 108, about 103 to about 107, about 104 to about 5 x 106 pfu or about 104 to about 1012 pfu. The dose of the pharmaceutical formulation to be administered to the subject may be 10 2, 5 x 102, 103, 5 x 103, 104, 5 x 104, 105, 5 x 105, 106, 5 x 106, 107, 5x 107, 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5x 1010, 1 x 1011, 5 x 1011 or 1012 pfu. 15 [0095] Applicants demonstrated that attenuated swine influenza viruses with impaired interferon antagonist activity were shown to replicate in vivo generating titers that are lower than than detected with wild-type swine influenza viruses, but that are sufficient to induce immunological and cytokine responses. Thus, mutations which diminish but do not abolish the IFN antagonist activity of the swine influenza virus are preferred for vaccine formulations. Such viruses can be selected for growth in both conventional and non-conventional substrates, and for intermediate virulence. 20 [0096] The present invention provides cells containing (alternatively, comprising) swine influenza viruses of the present invention. A cell may contain/comprise a swine influenza virus that is genetically engineered. The attenuated swine influenza virus contained in the cell may be engineered to encode an epitope derived from another virus or a tumor antigen. The genome of the attenuated swine influenza virus may comprise at least one segment derived from a different virus. Cells which can be infected with, contain or comprise an attenuated swine influenza virus of the invention include, 25 but are not limited to, MDCK cells, PK cells, Vero cells, primary human, umbilical vein endothelial cells (HUVEC), H292 human epithelial cell line, HeLa cells, and swine embryonic kidney cells RES. The cell may be a pig cell or a pig cell line. The cell (e.g., a pig cell or pig cell line) may be interferon deficient. [0097] The invention encompasses the use of substrates such as cells, cell lines and embryonated eggs, to propagate the attenuated swine influenza viruses of the invention. In one embodiment, the invention provides methods for vaccine 30 production, immunogenic formulation production and pharmaceutical production comprising propagating in a substrate an attenuated swine influenza virus of the present invention and collecting progeny virus, wherein the substrate is a cell, cell line or embryonated egg. The substrate may be an IFN deficient system. Exemplary IFN-deficient systems include, but are not limited to, young embryonated eggs (e.g., 6 to 10 days old, 6 to 9 days old, 6 to 8 days old or 6 to 7 days old embyronated eggs), IFN-deficient cell lines (such as VERO cells or genetically engineered cell lines such as STAT1 35 knockouts). Embryonated eggs or cell lines pretreated with compounds that inhibit the IFN system (including drugs, antibodies, antisense, ribozymes, etc.) can also be used as an IFN-deficient system. Further, eggs deficient in the IFN system, e.g., eggs produced by STAT1 negative birds, especially fowl, including but not limited to transgenic chickens, ducks or turkeys, may be used as an IFN-deficient system.

40 5.1 Generation of Mutants with Altered IFN Antagonist Activity

[0098] Any mutant swine influenza virus or strain which has a decreased IFN antagonist activity can be selected and used in accordance with the present disclosure, In one aspect, naturally occurring mutants or variants, or spontaneous swine influenza mutants can be selected that have an impaired ability to antagonize the cellular IFN response. In another 45 aspect, mutant swine influenza viruses can be generated by exposing the virus to mutagens, such as ultraviolet irradiation or chemical mutagens, or by multiple passages and/or passage in non-permissive hosts. Screening in a differential growth system can be used to select for those mutants having impaired IFN antagonist function. Since swine influenza virus A has a segmented genome, the attenuated phenotype can be transferred to another strain having a desired antigen by reassortment, ( i.e., by coinfection of the attenuated virus and the desired strain, and selection for reassortants 50 displaying both phenotypes). The swine influenza viruses of the invention are not naturally occuring viruses. The swine influenza viruses of the invention may be genetically engineered viruses. A swine influenza virus with naturally occurring mutations in the NS1 gene may be not encompassed by the invention. In certain aspects, known swine influenza viruses with mutations in the NS1 gene are not encompassed by the invention. In specific aspects, the swine influenza viruses disclosed herein contain all or a portion of the NS1 gene derived from human influenza viruses. 55 [0099] Mutations can be engineered into swine influenza virus using "reverse genetics" approaches. In this way, natural or other mutations which confer the attenuated phenotype can be engineered into vaccine strains. For example, deletions, insertions or substitutions of the coding region of the swine influenza virus gene for IFN antagonist activity ( i.e., the NS1 of swine influenza virus) can be engineered. Deletions, substitutions or insertions in the non-coding region of the swine

21 EP 2 497 492 B1

influenza virus gene responsible for IFN antagonist activity are also contemplated. To this end, mutations in the signals responsible for the transcription, replication, polyadenylation and/or packaging of the gene responsible or the IFN- antagonist activity can be engineered. Such mutations, for example to the promoter, could down-regulate the expression of the swine influenza virus gene responsible for IFN antagonist activity. Mutations in the promoter can be made, for 5 example, by promoter shuffling (e.g., of the influenza B virus promoter), or in the noncoding regions of the NS1 gene. Mutations in swine influenza virus genes which may regulate the expression of the swine influenza virus gene responsible for IFN antagonist activity (i .e., the swine influenza virus NS1 gene) are also within the scope of viruses that can be used in accordance with the invention. [0100] Disclosed herein are also swine influenza viruses comprising genomes comprising mutations to the NS 1 gene 10 segment that may not result in an altered IFN antagonist activity or an IFN-inducing phenotype but rather results in altered viral functions and an attenuated phenotype, e.g., altered inhibition of nuclear export of poly(A)-containing mRNA, altered inhibition of pre-mRNA splicing, altered inhibition of the activation of PKR by sequestering of dsRNA, altered effect on translation of viral RNA and altered inhibition of polyadenylation of host mRNA ( e.g., see Krug in Textbook of Influenza, Nicholson et al. Ed. 1998, 82-92, and references cited therein). 15 [0101] The reverse genetics technique involves the preparation of synthetic recombinant viral RNAs that contain the non-coding regions of the swine influenza virus RNA which are essential for the recognition by viral polymerases and for packaging signals necessary to generate a mature virion. The recombinant RNAs are synthesized from a recombinant DNA template and reconstituted in vitro with purified viral polymerase complex to form recombinant ribonucleoproteins (RNPs) which can be used to transfect cells. A more efficient transfection is achieved if the viral polymerase proteins 20 are present during transcription of the synthetic RNAs either in vitro or in vivo. The synthetic recombinant RNPs can be rescued into infectious virus particles. The foregoing techniques are described in U.S. Patent No. 5,166,057 issued November 24, 1992; in U.S. Patent No. 5,854,037 issued December 29, 1998; in European Patent Publication EP 0702085A1, published February 20,1996; in U.S. Patent Application Serial No. 09/152,845; in International Patent Publications PCT WO97/12032 published April 3,1997; WO96/34625 published November 7,1996; in European Patent 25 Publication EP-A780475; WO 99/02657 published January 21, 1999; WO 98/53078 published November 26, 1998; WO 98/02530 published January 22, 1998; WO 99/15672 published April 1, 1999; WO 98/13501 published April 2, 1998; WO 97/06270 published February 20, 1997; and EPO 780 475A1 published June 25, 1997. [0102] The helper-free plasmid technology can also be utilized to engineer an attenuated swine influenza virus. For a description of helper-free plasmid technology see, e.g., International Publication No. WO 01/04333; U.S. Patent No. 30 6,649,372; Fodor et al., 1999, J. Virol. 73:9679-9682; Hoffmann et al., 2000, Proc. Natl. Acad. Sci. USA 97:6108-6113; and Neumann et al., 1999, Proc. Natl. Acad. Sci. USA 96:9345-9350. [0103] Attenuated viruses generated by the reverse genetics approach or helper-free plasmid technology can be used in the vaccine, immunogenic and pharmaceutical formulations described herein. Reverse genetics techniques or helper- free plasmid technology can also be used to engineer additional mutations to other viral genes important for vaccine, 35 immunogenic and pharmaceutical formulation production-i.e., the epitopes of useful vaccine strain variants can be engineered into an attenuated swine influenza virus. Alternatively, completely foreign epitopes, including antigens derived from other viral or non-viral pathogens can be engineered into an attenuated strain. For example, antigens of non-related viruses, parasite antigens, bacterial or fungal antigens or tumor antigens can be engineered into an attenuated strain (e.g., epitopes of porcine reproductive and respiratory syndrome virus, porcine cytomegalo virus, porcine respiratory 40 corona virus, porcine encephalomyocarditis virus, porcine epidemic diarrhea and antigenic determinants of non-viral swine pathogens such as bacteria, including, but not limited to, Brucella suis, and parasites, including, but not limited to, roundworms ( Ascaris suum), whipworms ( Trichuris suis), or a tumor antigen such as carcinoembryonicantigen (CEA), breast cancer antigen such as EGFR (Epidermal growth factor receptor), HER2 antigen (p185 HER2), HER2 neu epitope, cancer antigen-50 (CA-50), cancer antigen 15-3 (CA15-3) associated with breast cancer, carcinoma associated antigen 45 (CAA), melanoma antigen, and melanoma associated antigens 100, 25, and 150). Alternatively, epitopes which alter the tropism of the virus in vivo can be engineered into the chimeric attenuated viruses disclosed herein. [0104] In a specific aspect, a combination of reverse genetics techniques or helper-free technology and reassortant techniques can be used to engineer attenuated viruses having the desired epitopes in swine influenza viruses. For example, an attenuated swine influenza virus (generated by, e.g., reverse genetics techniques, helper-free plasmid 50 technology or a combination thereof) and a strain carrying the desired vaccine epitope (generated by, e.g., natural selection, mutagenesis, by reverse genetics techniques, helper-free plasmid technology or a combination thereof) can be co-infected in hosts that permit reassortmentof the segmented genomes. Reassortants that display both the attenuated phenotype and the desired epitope can then be selected. Reassortment techniques can be used to transfer the attenuated phenotype from a parental swine influenze virus strain (a natural mutant, a mutagenized virus, or a genetically engineered 55 virus) to a different virus strain (a wild-type virus, a natural mutant, a mutagenized virus, or a genetically engineered virus ). [0105] In a specific aspect, disclosed herein is an attenuated swine influenza virus comprising a genome comprising a mutation in the swine influenza virus NS1 gene that diminishes the ability of the NS1 gene product to antagonize a cellular interferon response. In accordance with this aspect, the attenuated swine influenza virus is preferably genetically

22 EP 2 497 492 B1

engineered. [0106] The attenuated swine influenza viruses of the invention may have one or more or a combination of any of the following characteristics: the ability to induce interferon activity at a level less than (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% less than) that of wild-type swine 5 influenza virus, e.g., A/Swine/Texas/4199-2/98, as measured by standard interferon assays; a viral titer 1 to 50,2 to 25, 2 to 10 or 3 to 7 fold lower than wild-type swine influenza virus, e.g., A/Swine/Texas/4199-2/98, when grown under the same conditions (e.g., inoculated at a MOI of 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 or 10 and grown in PK cells, PK(D1) cells, PK(15) cells, PK13 cells, NSK cells, LLC-PK1 cells, LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells, PK-2a/CL 13 cells or SJPL cells and assayed in MDCK cells); a viral titer 1 to 10 fold, 1 to 5 fold, 5-10 10 fold, 10 to 500, 20 to 250, 20 to 100 or 40 to 80 fold lower than wild-type swine influenza virus, e.g., A/Swine/Tex- as/4199-2/98, when isolated from BALF of infected pigs as assayed in a plaque assay performed on MDCK cells; or a reduced ability to cause lung lesions in infected pigs.

aspect disclosed herein 15 [0107] In a specific aspect, an attenuated swine influenza virus disclosed herein has a genome comprising a mutation in a swine influenza virus NS1 gene that diminishes the ability of the NS1 gene product to antagonize a cellular interferon response, and permits the attenuated virus, at a multiplicity of infection (MOI) of between 0.0005 and 0.001, 0.001 and 0.01, 0.01 and 0.1, 0.1 and 1, or 0.1 and 1, or a MOI of 0.0005, 0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 20 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately 1 to approximately 100 fold, approximately 5 to approximately 80 fold, approximately 20 to approximately 80 fold, or approximately 40 to approximately 80 fold, approximately 1 to approximately 10 fold, approximately 1 to approximately 5 fold, approximately 1 to approximately 4 fold, approximately 1 to approximately 3 fold, approximately 1 to approximately 2 fold, or approximately 1, 2, 3, 4, 5, 6, 7, 8, or approximately 1, 2, 3, 4, 5, 7,10,15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold lower 25 than wild-type swine influenza virus in cells ( e.g., cells of a human (e.g., PerC6, a producer cell line derived from human embryonic retinoblasts transformed with the E1 region of Adenovirus 5), mouse, chicken (e.g., chicken embryo fibroblasts), rat, birds, or pig (e.g., PK(D1) cells, PK(15) cells, PK13 cells, NSK cells, LLC-PK1 cells, LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells, PK-2a/CL 13 or SJPL cells)) as determined by a hemagglutination assay of BALF obtained from pigs or supernatants of pig cells approximately 2 to 10 days, 3 to 7 days, 3 to 5 days or 2, 3, 5, 6, 7, 8, 9, 10 days 30 post-infection or when the viruses are plaqued on MDCK cells. The growth of an attenuated swine influenza virus may be compared to a particular standard or reference, e.g., wild-type swine influenza virus A/Swine/Texas/4199-2/98. The attenuated virus may be genetically engineered to contain or express non-swine influenza virus nucleic acid sequences such, e.g., an epitope of a foreign pathogen or a tumor antigen. Preferably, the non-swine influenza virus sequences do not include a nucleic acid sequence that alters the attenuated phenotype of the virus. Accordingly, nucleic acid 35 sequences encoding proteins, polypeptides or peptides with interferon antagonizing activity are preferably not engineered into swine influenza virus. [0108] Disclosed herein are attenuated swine influenza viruses comprising a genome comprising at least two, at least three, at least four or more mutations in two, three, four or more swine influenza virus genes, wherein at least one of the mutations is in the NS1 gene and contributes to or is responsible (directly or indirectly) for the attenuation of the 40 virus and/or the diminished ability of the virus to antagonize a cellular interferon response. In a specific aspect, an attenuated swine influenza virus comprises a genome comprising at least two, at least three, at least four or more mutations in two, three, four or more swine influenza virus genes, wherein at least one of the mutations is in the NS1 gene and is responsible for the diminished ability of the NS1 gene product to antagonize a cellular interferon response, and permits the attenuated virus, at a MOI of between 0.0005 and 0.001, 0.001 and 0.01,0.01 and 0.1, or 0.1 and 1, or 45 MOI of 0.0005, 0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately 1 to approximately 100 fold, approximately 5 to approximately 80 fold, approximately 20 to approximately 80 fold, or approximately 40 to approximately 80 fold, approximately 1 to approximately 10 fold, approximately 1 to approximately 5 fold, approximately 1 to approximately 4 fold, approximately 1 to approximately 3 fold, approximately 1 to approximately 2 fold, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 50 65, 70, 75, 80, 85, 90, 95 or 100 fold lower than wild-type swine influenza virus, e.g., A/Swine/Texas/4199-2/98, in pig cells, as determined by, e.g., hemagglutination assays, approximately 2 to 10 days, 3 to 7 days, or 2, 3, 5, 6, 7, 8, 9, 10 days post-infection when the viruses are propagated under the same conditions. In another aspect, an attenuated swine influenza virus disclosed herein comprises a genome comprising at least two, three, four or more mutations in two, three, four or more swine influenza virus genes, wherein at least one of the mutations is in the NS1 gene and is responsible 55 for the attenuation of the virus, and permits the attenuated virus, at a MOI of between 0.0005 and 0.001, 0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or a MOI of 0.0005, 0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately 1 to approximately 100 fold, approximately 5 to approximately 80 fold, approximately 20 to approximately 80 fold, or approximately 40 to approximately 80 fold, approximately 1 to

23 EP 2 497 492 B1

approximately 10 fold, approximately 1 to approximately 5 fold, approximately 1 to approximately 4 fold, approximately 1 to approximately 3 fold, approximately 1 to approximately 2 fold, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold lower than wild-type swine influenza e.g.,virus, A/Swine/Texas/4199-2/98, in pig cells, as determined by, e.g., hemagglutination assays, approximately 2 to 10 days, 3 5 to 7 days, 3 to 5 days, or 2, 3, 5, 6, 7, 8, 9, 10 days post-infection when the viruses are propagated, under the same conditions (e.g., in MDCK cells). In accordance with these aspects, the attenuated virus have an impaired interferon antagonist phenotype and the viruses may be genetically engineered to contain or express non-swine influenza virus nucleic acid sequences, such as, e.g., an epitope of a foreign pathogene.g., ( epitopes of porcine reproductive and respiratory syndrome virus, porcine cytomegalo virus, porcine respiratory corona virus, porcine encephalomyocarditis 10 virus, porcine epidemic diarrhea and antigenic determinants of non-viral pathogens such as bacteria and parasites, to name but a few) or a tumor antigen. Preferably, the non-swine influenza virus sequences (heterologous sequences) do not include a nucleic acid sequence that alters the attenuated phenotype of the virus. Accordingly, nucleic acid sequences encoding proteins, polypeptides or peptides with interferon antagonizing activity are preferably not engineered into the swine influenza virus. 15 [0109] Any mutation that results in the desired phenotype (preferably, an impaired ability to antagonize a cellular interferon response) can be introduced into the swine influenza virus NS1 gene. Examples of the types of mutations that can be included in or introduced into swine influenza virus NS1 gene include, but are not limited to, deletions, substitutions, insertions and combinations thereof. One or more mutations can be located anywhere throughout the NS1 gene (e.g., the amino-terminus, the carboxy-terminus or somewhere in between) and/or the regulatory element of the 20 NS1 gene. In a specific aspect, an attenuated swine influenza virus disclosed herein comprises a genome comprising a swine influenza virus NS1 gene with a mutation ( e.g., a deletion or substitution) at the amino-terminus. In a preferred aspect, an attenuated swine influenza virus disclosed herein comprises a genome comprising a swine influenza virus with a mutation (e.g., a deletion or substitution, preferably a deletion) at the carboxy-terminus. In another aspect, an attenuated swine influenza virus disclosed herein comprises a genome comprising a mutation in a swine influenza virus 25 NS1 gene resulting in a deletion consisting of 5, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 73, 75, 80, 85, 90, 95, 99, 100, 105, 110, 115, 120, 125, 126, 130, 135, 140, 145, 150, 155, 160, 165, 170 or 175 amino acid residues from the carboxy terminus of NS1, or a deletion of between 5-170,25-170, 50-170, 100-170, 100-160 or 105-160 amino acid residues from the carboxy- terminus. In another aspect, an attenuated swine influenza virus disclosed herein comprises a genome comprising a mutation in a swine influenza virus NS1 gene resulting in a deletion of all amino acid 30 residues except amino acid residues 1-126, amino acid residues 1-120, amino acid residues 1-115, amino acid residues 1-110, amino acid residues 1-100, amino acid residues 1-99, amino acid residues 1-95, amino acid residues 1-85, amino acid residues 1-80, amino acid residues 1-75, amino acid residues 1-73, amino acid residues 1-70, amino acid residues 1-65 or amino acid residues 1-60, wherein the amino terminal amino acid is number 1. In accordance with these aspects, , the attenuated swine influenza virus is preferably genetically engineered. An attenuated swine influenza virus of the 35 disclosure may be TX/98/del 126, TX/98/del 99 or TX/98/del 73. [0110] The attenuated swine influenza virus of the present invention may be a chimeric virus that expresses a heterologous sequence e.g., antigens of other vaccine strains e.g.( , using reverse genetics, reassortment or helper-free plasmid technology). Alternatively, the attenuated influenza viruses may be engineered, using reverse genetics, reassortment or helper-free plasmid technology with genetically engineered viruses, to express completely foreign epitopes, 40 e.g., antigens of other infectious pathogens, tumor antigens, or targeting antigens. The attenuated swine influenza viruses may express a heterologous sequence derived from other swine infectious agents, non-swine infectious agents, swine or other types of tumor antigens (e.g., carcinoembryonic antigen (CEA), breast cancer antigen such as EGFR (epidermal growth factor receptor), HER2 antigen (pl85HER2), HER2 neu epitope, cancer antigen-50 (CA-50), cancer antigen 15-3 (CA15-3) associated with breast cancer, carcinoma associated antigen (CAA), melanoma antigen, and 45 melanoma associated antigens 100, 25, and 150). The attenuated swine influenza virus may contain a segment derived from another virus. Since the NS RNA segment is the shortest among the eight viral RNAs, it is possible that the NS RNA will tolerate longer insertions of heterologous sequences than other viral genes. Moreover, the NS RNA segment directs the synthesis of high levels of protein in infected cells, suggesting that it would be an ideal segment for insertions of foreign antigens. Exemplary sequences include epitopes of porcine reproductive and respiratory syndrome virus, 50 porcine cytomegalo virus, porcine respiratory corona virus, porcine encephalomyocarditis virus, porcine epidemic diarrhea and antigenic determinants of non-viral swine pathogens such as bacteria, including, but not limited to, Brucella suis, and parasites, including, but not limited to, roundworms ( Ascaris suum), whipworms (Trichuris suis). [0111] Preferably, the heterologous sequence is not a nucleic acid sequence that alters the attenuated phenotype of the virus. Accordingly, preferably, nucleic acid sequences encoding proteins, polypeptides or peptides with interferon 55 antagonizing activity are not engineered into the swine influenza virus. The chimeric virus may express a tumor antigen. The chimeric virus may express an epitope of a foreign pathogen.

24 EP 2 497 492 B1

5.2 Selection of Attenuated Swine Influenza Viruses

[0112] Disclosed herein are methods of selecting swine influenza viruses which have the desired phenotype,i.e., attenuated swine influenza viruses or swine influenza viruses which have low IFN activity i.e.,( a reduction of 5-10%, 5 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or more when compared to wild-type swine influenza viruses, e.g., A/Swine/Texas/4199-2/98, under the same conditions) or no IFN antagonist activity whether obtained from natural variants, spontaneous variants (i.e., variants which evolve during virus propagation), mutagenized natural variants, reassortants and/or genetically engineered viruses (see, e.g., U.S. Patent No. 6,635,416). Such viruses can be best screened in differential growth assays that compare growth in IFN-deficient versus IFN-competent host 10 systems. Viruses which demonstrate better growth in the IFN-deficient systems versus IFN competent systems are selected; preferably, viruses which grow to titers at least one log, at least two logs, three logs, or 1 to 50, 1 to 25, 1 to 20, 1 to 10 or 1 to 5 logs greater in IFN-deficient systems as compared to an IFN-competent system are selected. Exemplary IFN-deficient systems include, but are not limited to, young embryonated eggs (e.g., 6 to 10 days old, 6 to 9 days old, 6 to 8 days old or 6 to 7 days old embryonated eggs), IFN-deficient cell lines (such as VERO cells or genetically 15 engineered cell lines such as STAT1 knockouts, PKR knockouts, etc.). Embryonated eggs or cell lines pretreated with compounds that inhibit the IFN system (including, drugs, antibodies, antisense, ribozymes, etc.) can also be used as an IFN-deficient system. Further, eggs deficient in the IFN system, e.g., eggs produced by STAT1 negative birds, especially fowl, including but not limited to transgenic chickens, ducks or turkeys, may be used as an IFN-deficient system. Attenuated swine influenza viruses showing at least 1 to 50, 1 to 25, 1 to 20, 1 to 10 or 1 to 5 logs lower titers 20 in 10-days-old eggs versus 6-7, 6-8, or 6-9 days old eggs will be considered impaired in their ability to inhibit the IFN response. [0113] For purposes of screening, transient IFN-deficient systems can be usedin lieu of genetically manipulated systems. For example, the host system can be treated with compounds that inhibit IFN production and/or components of the IFN response (e.g., drugs, antibodies against IFN, antibodies against IFN-receptor, inhibitors of PKR, antisense 25 molecules and ribozymes, etc.). Growth of attenuated swine influenza virus can be compared in IFN-competent untreated controls versus IFN-deficient treated systems. [0114] Growth of swine influenza virus (as measured by titer) can, e.g., be compared in a variety of cells, cell lines, or animal model systems that express IFN and the components of the IFN response, versus cells, cell lines, or animal model systems deficient for IFN or components of the IFN response. Techniques which are well known in the art for the 30 propagation of viruses in cell lines can be used (see, for example, the working examples infra). Growth of swine influenza virus in an IFN competent cell line versus an IFN deficient genetically engineered cell line can be compared. [0115] The swine influenza viruses can be screened using IFN assay systems e.g., transcription based assay systems in which reporter gene expression is controlled by an IFN-responsive promoter. Reporter gene expression in infected versus uninfected cells can be measured to identify viruses which efficiently induce an IFN response, but which are 35 unable to antagonize the IFN response. For example, test cells can be engineered to transiently or constitutively express reporter genes such as luciferase reporter gene or [0116] Chloramphenicol transferase (CAT) reporter gene under the control of an interferon stimulated response element, such as the IFN-stimulated promoter of the ISG-54K gene (Bluyssen et al., 1994, Eur. J. Biochem. 220:395-402). Cells are infected with the test swine influenza virus and the level of expression of the reporter gene compared to that 40 in uninfected cells or cells infected with wild-type swine influenza virus. An increase in the level of expression of the reporter gene following infection with the test attenuated swine influenza virus would indicate that the test swine influenza virus is inducing an IFN response. Alternatively, the induction of IFN responses may be determined by measuring IFN- dependent transcriptional activation following infection with the test attenuated swine influenza virus. The expression of genes known to be induced by IFN,e.g ., Mx, PKR, 2-5-oligoadenylatesynthetase, major histocompatibility complex 45 (MHC) class I, etc., can be analyzed by techniques known to those of skill in the art ( e.g., northern blots, western blots, PCR,etc.). The induction of IFN responses may also be determinedby measuring the phosphorylatedstate of components of the IFN pathway following infection with a test swine influenza virus, e.g., IRF-3, which is phosphorylated in response to double-stranded RNA. In response to type I IFN, Jak1 kinase and TyK2 kinase, subunits of the IFN receptor, STAT1, and STAT2 are rapidly tyrosine phosphorylated. Thus, in order to determine whether the swine influenza virus induces 50 IFN responses, cells, such as 293 cells, are infected with the test mutant virus and following infection, the cells are lysed. IFN pathway components, such as Jak1 kinase or TyK2 kinase, are immunoprecipitated from the infected cell lysates, using specific polyclonal sera or antibodies, and the tyrosine phosphorylated state of the kinase is determined by immunoblot assays with an anti-phosphotyrosine antibody ( e.g., see Krishnan et al. 1997, Eur. J. Biochem. 247: 298-305). An enhanced phosphorylated state of any of the components of the IFN pathway following infection with the swine 55 influenza virus would indicate induction of IFN responses by the swine influenza virus. [0117] Further, the induction of IFN responses following infection with a test swine infuenza virus may be determined by measuring the ability to bind specific DNA sequences or the translocation of transcription factors induced in response to viral infection, e.g., IRF3, STAT1, STAT2, etc. In particular, STAT1 and STAT2 are phosphorylated and translocated

25 EP 2 497 492 B1

from the cytoplasm to the nucleus in response to type I IFN. The ability to bind specific DNA sequences or the translocation of transcription factors can be measured by techniques known to those of skill in the art,e.g., electromobility gel shift assays, cell staining, etc. Other assays that can be used are described in U.S. Patent Application No. 09/829,711; In a preferred aspect, however, differential growth assays are used to select viruses having the desired phenotype, since 5 the host system used (IFN-competent versus IFN-deficient) applies the appropriate selection pressure. [0118] The attenuated swine influenza viruses of the present invention are optimally screened in pig cells, including primary, secondary pig cells and pig cell lines. Any pig cell which is capable of growing swine influenza virus can be used. Preferred pig cells include porcine kidney cell lines, porcine testis cells lines and porcine lung. Representative pig cells include, but are not limited to, PK(D1) cells, PK(15) cells, PK13 cells, NSK cells, LLC-PK1 cells, LLC-PK1A cells, 10 ESK-4 cells, ST cells, PT-K75 cells, PK-2a/CL 13 cells or SJPL cells. [0119] The swine influenza viruses of the invention can be screened in pig cells by comparison to wild-type swine influenza viruses, e.g., A/Swine/Texas/4199-2/98, under the same growth conditions. Attenuated swine influenza viruses are selected based on characteristics such as slower growth rates, fewer lung lesions upon infection of pigs, reduced viral titers in bronchoalveolar lavage fluid (BALF) and reduced detection of virus on nasal swabs when compared to wild- 15 type swine influenza virus. Such attenuated swine influenza viruses have decreased virulence because they have a reduced ability to replicate in interferon competent systems compared to wild-type swine influenza viruses but can generate an immune response. [0120] Pig cells can be infected with wild-type swine influenza viruses, e.g., A/Swine/Texas/4199-2/98, and NS1 mutants at a specific MOI and viral titers in the supernatant can be determined at specific times post-infection. Infection 20 of pig cells at MOIs from between 0.0005 and 0.001, 0.001 and 0.01, 0.01 and 0.1, 0.1 and 1, or 1 and 10, or a MOI of 0.0005, 0.001, 0.005 0.01, 0.05, 0.1, 0.5, 1,5 or 10 can be used. In one embodiment, an MOI of 0.001 is used. Viral titers can be determined from approximately 2 to 10 days, 3 to 7 days, 3 to 5 days or 2, 3, 4, 5, 6, 7, 8, 9 or 10 days post-infection. In a specific aspect, viral titers are determined 5 days post-infection. After growing virus in pig cells, vira l titers can be assessed in the supernatant. Any system for measuring titers of influenza virus can be used. Representative 25 systems are described in Section 5.7. In a particular aspect, viral titers in the supernatant are determined by plaquing various time points on MDCK cells. [0121] The selection systems disclosed herein encompass measuring IFN induction by determining whether an extract from the cell or egg infected with the test attenuated swine influenza virus is capable of conferring protective activity against viral infection. More specifically, groups of 10-day old embryonated chicken eggs are infected with the test mutant 30 swine influenza virus or the wild-type swine influenza virus. Approximately 15 to 20 hours post infection, the allantoic fluid is harvested and tested for IFN activity by determining the highest dilution with protective activity against swine influenza virus infection in tissue culture cells. [0122] The pathogenesis of mutant swine influenza viruses of the invention can also be assessed in pigs in vivo. Useful assays include assessing lung lesions in lung lobes, nasal swabs and determination of viral titers in bronchoalveolar 35 lavage fluid (BALF) by any method known in the art.

5.3 Propagation of Attenuated Swine Influenza Virus

[0123] Disclosed herein are methods for propagating attenuated swine influenza viruses in cells (e.g., pig cells), 40 embryonated eggs, and animals. The attenuated swine influenza viruses of the present invention can be propagated in any substrate that allows the virus to grow to titers that permit a use of the attenuated swine influenza virus described herein. The attenuated swine influenza viruses of the present invention may be propagated in any substrate that allows the virus to grow to titers comparable to those determined for wild type swine influenza virus strains in IFN-competent substrates. The attenuated swine influenza viruses of the invention may be propagated in IFN-deficient substrates. 45 Substrates which are useful for selection of the attenuated swine influenza viruses of the invention do not have to be used for propagation and vice versa. [0124] In accordance with the methods disclosed herein, attenuated swine influenza viruses which may be grown in cells (e.g., pig cells), embryonated eggs, and animals are selected from naturally occurring strains, variants or mutants, mutagenized virus, reassortants and/or genetically engineered viruses. The methods disclosed herein encompass grow- 50 ing the attenuated swine influenza viruses, preferably using appropriate growth conditions (see e.g., growth conditions set forth in Section 6 below), and collecting the progeny virus. [0125] The attenuated swine influenza viruses of the invention may be propagated in pig cells. The pig cells may or may not be IFN-deficient or produce lower levels of IFN. Preferred pig cells include porcine kidney cell lines, porcine testis cell lines and porcine lung cell lines. Representative pig cells include, but are not limited to, PK(D1) cells, PK(15) 55 cells, PK13 cells, SJPL cells, NSK cells, LLC-PK1 cells, LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells, and PK- 2a/CL 13 cells. In another specific aspect, the attenuated swine influenza viruses are propagated in chicken cells, e.g., chicken embryo fibroblasts derived from 6-day-old embryonated eggs. [0126] In certain aspects, disclosed herein are methods of propagating the attenuated swine influenza viruses disclosed

26 EP 2 497 492 B1

herein in embryonated eggs, e.g., from 6 to 14 days old. In other aspects, young or immature embryonated eggs can be used to propagate attenuated swine influenza viruses of the invention. Immature embryonated eggs encompass eggs which are less than ten-day-old eggs, preferably six to nine day old eggs, six to eight day old, six to seven day old eggs or six days old eggs. Immature embryonated eggs also encompass eggs which artificially mimic immature eggs up to, 5 but less than ten-day-old, as a result of alterations to the growth conditions, e.g., changes in incubation temperatures; treating with drugs; or any other alteration which results in an egg with a retarded development, such that the IFN system is not fully developed as compared with ten-to twelve-day-old eggs. The swine influenza viruses can be propagated in different locations of the embryonated egg, e.g., the allantoic cavity. The embryonated eggs may be chick eggs. [0127] The disclosure also encompasses methods and IFN-deficient substrates for the growth and isolation of atten- 10 uated swine influenza viruses. See, e.g., U.S. Patent No. 6,573,079. IFN-deficient substrates which can be used to support the growth of attenuated swine influenza viruses include, but are not limited to, naturally occurring cells, cell lines, embryonated eggs, and IFN-deficient systems, e.g., Vero cells, young embryonated eggs; recombinant cells or cell lines that are engineered to be IFN deficient, e.g., IFN-deficient cell lines derived from STAT1 knockouts, IRF3 knockouts, IRF7 knockouts, PKR knockouts, etc.; embryonated eggs obtained from IFN deficient birds, especially fowl 15 (e.g., chickens, ducks, turkeys) including flock that are bred to be IFN-deficient or transgenic birds (e.g., STAT1 knockouts). In certain aspects, the IFN-deficient substrate is not Vero cells and/or not a STAT1 deficient cell line. [0128] The host system, cells, cell lines, eggs or animals can be genetically engineered to express transgenes encoding inhibitors of the IFN system, e.g., dominant-negative mutants, such as STAT1 lacking the DNA binding domain, antisense RNA, ribozymes, inhibitors of IFN production, inhibitors of IFN signaling, and/or inhibitors of antiviral genes induced by 20 IFN. It should be recognized that animals that are bred or genetically engineered to be IFN deficient will be somewhat immunocompromised, and should be maintained in a controlled, disease free environment. Thus, appropriate measures (including the use of dietary antibiotics) should be taken to limit the risk of exposure to infectious agents of transgenic IFN deficient animals, such as mice, flocks of breeding hens, ducks, turkeys etc. Alternatively, the host system, e.g., cells, cell lines, eggs or animals can be treated with a compound which inhibits IFN production and/or the IFN pathway 25 e.g., drugs, antibodies, antisense molecules, ribozyme molecules targeting the STAT1 gene, and/or antiviral genes induced by IFN. [0129] Disclosed herewith are methods of growing and isolating swine influenza viruses having altered IFN antagonist activity in cells and cell lines which naturally do not have an IFN pathway or have a deficient IFN pathway or have a deficiency in the IFN system e.g., low levels of IFN expression as compared to wild-type cells. In a particular aspect, 30 disclosed herein are methods of growing the attenuated swine influenza viruses of the invention in chicken embryo fibroblasts derived from 6-day-old embryonated eggs, or Vero cells, or IFN-compromised embryonated eggse.g. ( , immature embryonated eggs such as 6-, 7- or 8-day old embryonated eggs). In another aspect, disclosed herein are methods of growing attenuated swine influenza viruses in cells where the cells are not Vero cells. [0130] Disclosed herein are methods of growing and isolating swine influenza viruses from a genetically engineered 35 IFN deficient substrate. Disclosed herein are transgenic pigs and avians in which a gene essential to the IFN system is mutated, e.g., STAT1, which would lay eggs that are IFN deficient. Further, disclosed herein are avian transgenics which express dominant-negative transcription factors, e.g., STAT1 lacking the DNA binding domain, ribozymes, antisense RNA, inhibitors of IFN production, inhibitors of IFN signaling, and inhibitors of antiviral genes induced in response to IFN. [0131] Disclosed herein are recombinant cell lines or animals, in particular pigs and avians, in which one or more 40 genes essential for IFN synthesis, the IFN pathway, and/or an antiviral gene induced by IFN,e.g. interferon receptor, STAT1, PKR, IRF3, IRF7, etc. has been mutated ( e.g., disrupted, i.e., is a "knockout"). The recombinant animal can be any animal (such as a mouse, a pig or an avian,e.g., chicken, turkey, hen, duck, etc. (see, e.g.; Sang, 1994, Trends Biotechnol. 12:415; Perry, et al., 1993, Transgenic Res. 2:125; Stern, C.D., 1996, Curr Top Microbiol Immunol 212:195-206; and Shuman, 1991, Experientia 47;897 for reviews regarding the production of avian transgenics)). In a 45 specific aspect, the recombinant animal is a pig. Such a cell line or animal can be generated by any method known in the art for disrupting a gene on the chromosome of the cell or animal. Such techniques include, but are not limited to pronuclear microinjection (Hoppe & Wagner, 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson et al., 1989, Cell 56:313); electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803); and sperm- 50 mediated gene transfer (Lavitrano et al.; 1989, Cell 57:717); etc. For a review of such techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115:171. [0132] In particular, a STAT1 knockout animal can be produced by promoting homologous recombination between a STAT1 gene in its chromosome and an exogenous STAT1 gene that has been rendered biologically inactive (preferably by insertion of a heterologous sequence, e.g, an antibiotic resistance gene). Homologous recombination methods for 55 disrupting genes in the mouse genome are described, for example, in Capecchi (1989, Science 244:1288) and Mansour et al. (1988, Nature 336:348-352). [0133] Briefly, all or a portion of a STAT1 genomic clone is isolated from genomic DNA from the same species as the knock-out cell or animal. The STAT1 genomic clone can be isolated by any method known in the art for isolation of

27 EP 2 497 492 B1

genomic clones (e.g. by probing a genomic library with a probe derived from a STAT1 sequence such as pig STAT1 (Genbank Accession Nos. AB116564 and NM_213769) and those sequences provided in see Meraz et al. 1996, Cell 84: 431-442; Durbin et al. 1996, Cell 84: 443-450, and references cited therein). Once the genomic clone is isolated, all or a portion of the clone is introduced into a recombinant vector. Preferably, the portion of the clone introduced into the 5 vector contains at least a portion of an exon of the STAT1 gene,i.e ., contains a STAT1 protein coding sequence. A sequence not homologous to the STAT1 sequence, preferably a positive selectable marker, such as a gene encoding an antibiotic resistance gene, is then introduced into the STAT1 gene exon. The selectable marker is preferably operably linked to a promoter, more preferably a constitutive promoter. The non-homologous sequence is introduced anywhere in the STAT1 coding sequence that will disrupt STAT1 activity, e.g., at a position where point mutations or other mutations 10 have been demonstrated to inactivate STAT1 protein function. For example, but not by way of limitation, the non- homologous sequence can be inserted into the coding sequence for the portion of the STAT1 protein containing all or a portion of the kinase domain (e.g., the nucleotide sequence coding for at least 50, 100, 150, 200 or 250 amino acids of the kinase domain). [0134] The positive selectable marker is preferably a neomycin resistance gene ( neo gene) or a hygromycin resistance 15 gene (hygro gene). The promoter may be any promoter known in the art; by way of example the promoter may be the phosphoglycerate kinase (PGK) promoter (Adra et al., 1987, Gene 60:65-74), the PolII promoter (Soriano et al., 1991, Cell 64:693-701), or the MC1 promoter, which is a synthetic promoter designed for expression in embryo-derived stem cells (Thomas & Capecchi, 1987, Cell 51:503-512). Use of a selectable marker, such as an antibiotic resistance gene, allows for the selection of cells that have incorporated the targeting vector (for example, the expression of the neo gene 20 product confers resistance to G418, and expression of the hygro gene product confers resistance to hygromycin). [0135] A negative selectable marker for a counterselection step for homologous, as opposed to non-homologous, recombination of the vector may be inserted outside of the STAT1 genomic clone insert. For example, such a negative selectablemarker is theHSV thymidine kinase gene (HSV- tk), the expression of which makes cells sensitive to ganciclovir. The negative selectable marker is preferably under the control of a promoter such as, but not limited to the PGK promoter, 25 the PolII promoter or the MC1 promoter. [0136] When homologous recombination occurs, the portions of the vector that are homologous to the STAT1 gene, as well as the non-homologous insert within the STAT1 gene sequences, are incorporated into the STAT1 gene in the chromosome, and the remainder of the vector is lost. Thus, since the negative selectable marker is outside the region of homology with the STAT1 gene, cells in which homologous recombination has occurred (or their progeny), will not 30 contain the negative selectable marker. For example, if the negative selectable marker is the HSV- tk gene, the cells in which homologous recombination has occurred will not express thymidine kinase and w ill survive exposure to ganciclovir. This procedure permits the selection of cells in which homologous recombination has occurred, as compared to non- homologous recombination in which it is likely that the negative selectable marker is also incorporated into the genome along with the STAT1 sequences and the positive selectable marker. Thus, cells in which non-homologous recombination 35 has occurred would most likely express thymidine kinase and be sensitive to ganciclovir. [0137] Once the targeting vector is prepared, it is linearized with a restriction enzyme for which there is a unique site in the targeting vector, and, the linearized vector is introduced into embryo-derived stem (ES) cells (Gossler et al., 1986, Proc. Natl. Acad. Sci. USA 83:9065-9069) by any method known in the art, for example by electroporation. If the targeting vector includes a positive selectable marker and a negative, counterselectable marker, the ES cells in which homologous 40 recombination has occurred can be selected by incubation in selective media. For example, if the selectable markers are the neo resistance gene and the HSV-tk gene, the cells are exposed to G418 (e.g., approximately 300 mg/ml) and ganciclovir (e.g., approximately 2 mM). [0138] Any technique known in the art for genotyping, for example but non limited to Southern blot analysis or the polymerase chain reaction, can be used to confirm that the disrupted STAT1 sequences have homologously recombined 45 into the STAT1 gene in the genome of the ES cells. Because the restriction map of the STAT1 genomic clone is known and the sequence of the STAT1 coding sequence is known (see Meraz et al. 1996, Cell 84:431, Durbin et al. 1996, Cell 84:443-450, all references cited therein), the size of a particular restriction fragment or a PCR amplification product generated from DNA from both the disrupted and non-disrupted alleles can be determined. Thus, by assaying for a restriction fragment or PCR product, the size of which differs between the disrupted and non-disrupted STAT1 gene, 50 one can determine whether homologous recombination has occurred to disrupt the STAT1 gene. [0139] The ES cells with the disrupted STAT1 locus can then be introduced into blastocysts by microinjection and then the blastocysts can be implanted into the uteri of pseudopregnant mice using routine techniques. The animal that develop from the implanted blastocysts are chimeric for the disrupted allele. The chimeric males can be crossed to females, and this cross can be designed such that germline transmission of the allele is linked to transmission of a 55 certain coat color. The germline transmission of the allele can be confirmed by Southern blotting or PCR analysis, as described above, of genomic DNA isolated from tissue samples. [0140] Any gene whose product is important for interferon regulation can be used. Other mutantions in the interferon pathway which may be used in accordance with the present disclosure include kinase deficient versions of Jak1, TyK2

28 EP 2 497 492 B1

or transcription factors lacking DNA binding domains STAT1, and STAT2 (see,e.g., Krishnan et al., 1997, Eur. J. Biochem. 247: 298-305). [0141] For virus purification, the attenuated swine influenza virus is removed from cell culture and separated from cellular components, typically by well known clarification procedures, e.g., such as gradient centrifugation and column 5 chromatography, and may be further isolated as desired using procedures well known to those skilled in the art, e.g., plaque assays.

5.4 Uses of Attenuated Viruses

10 [0142] Attenuated swine influenza viruses of the invention can be used as viral vectors for production of heterologous proteins as described in U.S. Patent No. 5,820,871 . [0143] Attenuated swine influenza viruses of the invention can be used in screening assays to identify viral proteins with interferon antagonizing function, for identifying viral proteins that have the ability to complement replication of an attenuated virus with impaired ability to antagonize cellular interferon responses and screening assays to identify anti- 15 viral agents which inhibit interferon antagonist activity and inhibit viral replication as described in U.S. Patent No. 6,635,416. [0144] Attenuated swine influenza viruses of the invention can be used to produce antibodies which can be used in diagnostic immunoassays, passive immunotherapy, and generation of antiidiotypic antibodies. For example, an attenuated swine influenza virus comprising a genome comprising a mutation in the NS1 gene and a heterologous sequence 20 (e.g., a tumor antigen) can be administered to a subject ( e.g., a pig) to generate antibodies which can then be isolated and used in diagnostic assays, passive immunotherapy and generation of antiidiotypic antibodies. The generated antibodies may be isolated by standard techniques known in the art (e.g., immunoaffinity chromatography, centrifugation, precipitation, etc.) and used in diagnostic immunoassays, passive immunotherapy and generation of antiidiotypic antibodies. The isolated antibodies before being used in passive immunotherapy may be modified, e.g., the antibodies may 25 be chimerized or humanized. See, e.g., U.S. Patent Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, for reviews on the generation of chimeric and humanized antibodies. [0145] The antibodies isolated from subjects administered an attenuated swine influenza virus of the invention may also be used to monitor treatment and/or disease progression. Any immunoassay system known in the art may be used 30 for this purpose including but not limited to competitive and noncompetitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme-linked immunosorbent assays), "sandwich" immunoassays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immuno- radiometric assays, fluorescent immunoassays, protein A immunoassays and immunoelectrophoresis assays, to name but a few. 35 [0146] The antibodies generated by the attenuated swine influenza viruses of the invention can also be used in the production of antiidiotypic antibody. The antiidiotypic antibody can then in turn be used for immunization, in order to produce a subpopulation of antibodies that bind the initial antigen of the pathogenic microorganism (Jerne, 1974, Ann. Immunol. (Paris) 125c:373; Jerne et al., 1982, EMBO J. 1:234). [0147] In immunization procedures, the amount of immunogen to be used and the immunization schedule will be 40 determined by a physician skilled in the art and will be administered by reference to the immune response and antibody titers of the subject.

5.5 Vaccine Formulations/Immunogenic Formulations

45 [0148] Disclosed herein are vaccine formulations and immunogenic formulations comprising an attenuated swine influenza virus having an impaired ability to antagonize the cellular IFN responsei.e., ( swine influenza viruses with mutations in the NS1 gene that impair the ability of the virus to induce a cellular IFN response), and a suitable excipient. The attenuated swine influenza virus used in the vaccine formulation or immunogenic formulation may be selected from naturally occurring mutants or variants, mutagenized viruses or genetically engineered viruses. The attenuated swine 50 influenza virus may be genetically engineered. The attenuated swine influenza virus may be isolated or purified. Atten- uated strains of swine influenza viruses can also be generated via reassortment techniques, helper-free plasmid technology or by using a combination of the reverse genetics approach or helper-free plasmid technology and reassortment techniques. Naturally occurring variants include viruses isolated from nature as well as spontaneous occurring variants generated during virus propagation, having an impaired ability to antagonize the cellular IFN response. The attenuated 55 swine influenza virus can itself be used as the active ingredient in the vaccine formulation or immunogenic formulation. Alternatively, the attenuated swine influenza virus can be used as the vector or "backbone" of recombinantly produced vaccines or immunogenic formulations. To this end, recombinant techniques such as reverse genetics (or, for segmented viruses, combinations of the reverse genetics and reassortment techniques) may be used to engineer mutations or

29 EP 2 497 492 B1

introduce foreign antigens into the attenuated swine influenza virus used in the vaccine formulation or immunogenic formulation. In this way, vaccines can be designed for immunization against strain variants, or in the alternative, against completely different infectious agents or disease antigens. [0149] Virtually heterologous gene sequence may be constructed into the attenuated swine influenza viruses of the 5 invention for use in vaccines or immunogenic formulations. Preferably, epitopes that induce a protective immune response to any of a variety of pathogens, or antigens that bind neutralizing antibodies may be expressed by or as part of the viruses. For example, heterologous gene sequences that can be constructed into the viruses of the invention for use in vaccines and immunogenic formulations include but are not limited to antigenic determinants of non-viral pathogens such as bacteria and parasites. All or portions of immunoglobulin genes may be expressed. For example, variable regions 10 of anti-idiotypic immunoglobulins that mimic such epitopes may be constructed into the viruses of the invention. Tumor associated antigens may be expressed. Examples of tumor antigens include, but are not limited to, KS 1/4 pan-carcinoma antigen, ovarian carcinoma antigen (CA125), prostatic acid phosphate, prostate specific antigen, melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate specific membrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen, milk fat globule antigen, colorectal 15 tumor-associated antigens (such as: CEA, TAG-72, C017-1A; GICA 19-9, CTA-1 and LEA), Burkitt’s lymphoma antigen- 38.13, CD19, B-lymphoma antigen-CD20, CD33, melanoma specific antigens (such as ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3), tumor-specific transplantation type of cell-surface antigen (TSTA) (such as virally-induced tumor antigens including T-antigen DNA tumor viruses and Envelope antigens of RNA tumor viruses), oncofetal antigen-alpha-fetoprotein such as CEA of colon, bladder tumor oncofetal antigen, differentiation antigen (such 20 as human lung carcinoma antigen L6 and L20), antigens of fibrosarcoma, leukemia T cell antigen-Gp37, neoglycoprotein, sphingolipids, breast cancer antigens (such as EGFR (Epidermal growth factor receptor), HER2 antigen (p185 HER2) and HER2 neu epitope), polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen-APO-1, differentiation antigen (such as I antigen found in fetal erythrocytes, primary endoderm, I antigen found in adult erythrocytes, preim- plantation embryos, I(Ma) found in gastric adenocarcinomas, M18, M39 found in breast epithelium, SSEA-1 found in 25 myeloid cells, VEP8. VEP9, Myl, VIM-D5, D156-22 found in colorectal cancer, TRA-1-85 (blood group H), C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten, Ley found in embryonal carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells , E1 series (blood group B) found in pancreatic cancer, FC10.2 found in embryonal carcinoma cells, gastric adenocarcinoma antigen, CO-514 (blood group Lea) found in Adenocarcinoma, NS-10 found in adenocarcinomas, CO-43 (blood group Le b), G49 found in EGF receptor 30 of A431 cells, MH2 (blood group ALe b/Ley) found in colonic adenocarcinoma, 19.9 found in colon cancer, gastric cancer mucins, T5A7 found in myeloid cells, R 24 found in melanoma, 4.2, G D3, D1.1, OFA-1, GM2, OFA-2, GD2, and M1:22:25:8 found in embryonal carcinoma cells, and SSEA-3 and SSEA-4 found in 4 to 8-cell stage embryos), T cell receptor derived peptide from a Cutaneous T cell Lymphoma, C-reactive protein (CRP), cancer antigen-50 (CA-50), cancer antigen 15-3 (CA15-3) associated with breast cancer, cancer antigen-19 (CA-19) and cancer antigen-242 associated with gastroin- 35 testinal cancers, carcinoma associated antigen (CAA), chromogranin A, epithelial mucin antigen (MC5), human epithelium specific antigen (HEA), Lewis(a)antigen, melanoma antigen, melanoma associated antigens 100, 25, and 150, mucin-like carcinoma-associated antigen, multidrug resistance related protein (MRPm6), multidrug resistance related protein (MRP41), Neu oncogene protein (C-erbB-2), neuron specific enolase (NSE), P-glycoprotein (mdr1 gene product), multidrug-resistance-related antigen, p170, multidrug-resistance-related antigen, prostate specific antigen (PSA), CD56, 40 and NCAM. [0150] Either a live recombinant swine influenza virus vaccine or immunogenic formulation or an inactivated recombinant swine influenza virus vaccine or immunogenic formulation can be formulated. A swine influenza virus can be inactivated by methods well known to those of skill in the art. Common methods use formalin and heat for inactivation. See, e.g., U.S. Patent No. 6,635,246. Other methods include those described in U.S. Patent Nos. 5,891,705; 5,106,619 45 and 4,693,981. [0151] A live vaccine or immunogenic formulation may be preferred because multiplication in the host leads to a prolonged stimulus of similar kind and magnitude to that occurring in natural infections, and therefore, confers substantial, long-lasting immunity. Production of such live recombinant swine influenza virus vaccine formulations and immunogenic formulations may be accomplished using conventional methods involving propagation of swine influenza virus in cell 50 culture or in the allantois of the chick embryo followed by purification. Moreover, the attenuated swine influenza viruses can induce a robust IFN response which has other biological consequences in vivo, affording protection against subsequent infectious diseases and/or inducing antitumor responses. [0152] Vaccine formulations and immunogenic formulations may include genetically engineered swine influenza influenza viruses that have mutations in the NS1 gene including but not limited to the truncated NS1 influenza mutants 55 described in the working examples, infra. They may also be formulated using natural variants. When formulated as a live virus vaccine, a range of about 102 to 108 can be used, preferably from about 103 to 107, more preferably 104 pfu to about 5x106, and most preferably from 104 to 107 pfu per dose should be used. [0153] Swine influenza virus may contain mutations to the NS1 gene segment that may not result in an altered IFN

30 EP 2 497 492 B1

antagonist activity or an IFN-inducing phenotype but rather result in altered viral functions and an attenuated phenotype e.g., altered inhibition of nuclear export of poly(A)-containing mRNA, altered inhibition of pre-mRNA splicing, altered inhibition of the activation of PKR by sequestering of dsRNA, altered effect on translation of viral RNA and altered inhibition of polyadenylation of host mRNA (e.g., see Krug in Textbook of Influenza, Nicholson et al. Ed. 1998, 82-92, 5 and references cited therein). [0154] Many methods may be used to introduce the vaccine formulations and immunogenic formulations described above, these include but are not limited to intranasal, intratracheal oral, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. It may be preferable to introduce the swine influenza virus vaccine formulation or immunogenic formulation via the natural route of infection of the pathogen for which the vaccine is designed, or via 10 the natural route of infection of the wild-type virus. Where a live attenuated swine influenza virus vaccine preparation or immunogenic formulation is used, it may be preferable to introduce the formulation via the natural route of infection for influenza virus. The ability of influenza virus to induce a vigorous secretory and cellular immune response can be used advantageously. For example, infection of the respiratory tract by attenuated swine influenza viruses may induce a strong secretory immune response, for example in the urogenital system, with concomitant protection against a particular 15 disease causing agent. [0155] In a specific aspect, it may be desirable to administer the compositions disclosed herein locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one aspect, administration can be by direct 20 injection at the site (or former site) or infection. [0156] A vaccine or immunogenic formulation disclosed herein, comprising 10 2 to 108 pfu, preferably from about 103 to 107, more preferably 10 4 pfu to about 5x10 6 pfu of attenuated swine influenza virus with altered IFN antagonist activity, could be administered once to a subject. Alternatively, a vaccine or immunogenic formulation disclosed herein, comprising 102 to 108, preferably from about 103 to 107, more preferably 104 to about 5x106 pfu of mutant viruses with altered IFN 25 antagonist activity, could be administered twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times to a subject with an interval of 2 to 6 months, 2 to 8 months, 2 to 10 months, or 2 to 12 months between doses. Alternatively, a vaccine or immunogenic formulation disclosed herein, comprising 10 2 to 108, preferably from about 103 to 107, more preferably 10 4 pfu to about 5x10 6 pfu of mutant viruses with altered IFN antagonist activity, could be administered as often as needed to a subject. The subject may be pig. 30 [0157] A vaccine formulation or an immunogenic formulation may not result in complete protection from an infection (e.g., a viral infection), but results in a lower titer or reduced number of the pathogen (e.g., a virus) compared to an untreated subject. Benefits include, but are not limited to, less severity of symptoms of the infection and a reduction in the length of the disease or condition associated with a the infection [0158] The vaccine formulation or immunogenic formulation of the invention may be used to protect against infections 35 by swine influenza virus. In other aspects, a vaccine formulation disclosed herein is used to protect against infection by another swine virus, including, but not limited to, porcine reproductive and respiratory syndrome virus, porcine cytomegalo virus, porcine respiratory corona virus, porcine encephalomyocarditis virus, porcine epidemic diarrhea. The vaccine formulation or immunogenic formulation of the invention may be used to protect against infections by pathogens other than a swine virus. 40 [0159] An immunogenic formulation of the invention may be used for preventing, treating, managing or ameliorating conditions in which the induction of an immune response to a particular antigen is beneficial.

5.6 Pharmaceutical Formulations

45 [0160] The present invention encompasses pharmaceutical formulations comprising swine influenza viruses of the present invention with altered IFN antagonist activity to be used as anti-viral agents or anti-tumor agents or as agents against IFN-treatable diseases. The pharmaceutical formulations may have utility as an anti-viral prophylactic and may be administered to a pig at risk of getting infected or is expected to be exposed to a virus, e.g., swine influenza virus. The pharmaceutical formulations may have utility as a therapeutic or prophylatic for an IFN-treatable disease and thus, 50 can be used for treating; preventing, managing or ameliorating an IFN-treatable disorders. The pharmaceutical formulations may have utility as a therapeutic or prophylatic for a swine IFN-treatable disease and thus, can be used for treating, preventing, managing or ameliorating a swine. IFN-treatable disorders. In specific aspects, the pharmaceutical formulations disclosed herein have utility as a therapeutic or prophylactic for an IFN-treatable disease and thus, can be used for treating, preventing, managing or ameliorating an IFN-treatable disorders in donkeys, zebras, camels, dogs, 55 avians (e.g., ducks). The pharmaceutical formulations of the invention may have utility as a therapeutic or prophylactic for an IFN-treatable disease and thus, can be used for treating, preventing, managing or ameliorating an IFN-treatable disorders in pigs. [0161] A pharmaceutical formulation of the invention may comprise a chimeric attenuated swine influenza virus of the

31 EP 2 497 492 B1

present invention comprising a heterologous sequence. The heterologous sequence can encode an epitope from a foreign or tumor antigen. A foreign antigen can be selected from another virus, a bacteria or a parasite. The pharmaceutical formulations may be used to treat tumors or prevent tumor formation, e.g., in pigs who have cancer or in those who are at high risk for developing neoplasms or cancer. For example, a subject e.g.,( a pig, donkey, or other animal that can 5 be infected with swine influenza virus or in which swine influenza virus can replicate) with cancer can be treated to prevent further tumorigenesis. Alternatively, pigs who are or are expected to be exposed to carcinogens can be treated. Alternatively, pigs who are to be exposed to radiation can be treated prior to exposure and thereafter. Specific cancers that can be treated with the compostions of the present invention include, but are not limited to, cancers of the uterus, mammary gland, pancreas, skin, lymph gland, vulva and testicle. viruses and formulations of the 10 [0162] The antitumor properties of the viruses and formulations of the invention can be at least partially related to their ability to induce IFN and IFN responses. Alternatively, the antitumor properties of the attenuated swine influenza viruses of the invention can be related to their ability to specifically grow in and kill tumor cells, many of which are known to have deficiencies in the IFN system. Regardless of the molecular mechanism(s) responsible for the antitumor properties, the attenuated swine influenza viruses of the invention may be used to treat tumors or to prevent tumor formation. 15 [0163] Disclosed herein are the swine influenza viruses with an altered IFN-antagonist phenotype which are targeted to specific organs, tissues and/or cells in the body in order to induce therapeutic or prophylactic effects locally. One advantage of such an approach is that the IFN-inducing swine influenza viruses disclosed herein are targeted to specific sites, e.g. the location of a tumor, to induce IFN in a site specific manner for a therapeutic effect rather than inducing IFN systemically which may have toxic effects. 20 [0164] The IFN-inducing swine influenza viruses disclosed herein may be engineered using the methods described herein to express proteins or peptides which would target the viruses to a particular site. In a specific aspect, the IFN- inducing swine influenza viruses would be targeted to sites of tumors. In such aspect, the swine influenza viruses can be engineered to express the antigen combining site of an antibody which recognizes a tumor specific antigen, thus targeting the IFN-inducing swine influenza virus to the tumor. In another aspect, where the tumor to be targeted expresses 25 a hormone receptor, such as breast or ovarian tumors which express estrogen receptors, the IFN-inducing swine influenza virus may be engineered to express the appropriate hormone. In yet another aspect, where the tumor to be targeted expresses a receptor to a growth factor, e.g. VEGF, EGF, or PDGF, the IFN-inducing virus may be engineered to express the appropriate growth factor or fragment thereof. Thus, in accordance with the disclosure, the IFN-inducing swine influenza viruses may be engineered to express any target gene product, including peptides, proteins, such as enzymes, 30 hormones, growth factors, antigens or antibodies, which w ill function to target the swine influenza virus to a site in need of treatment (e.g., anti-viral, antibacterial, anti-microbial or anti-cancer therapy). [0165] Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The pharmaceutical formulations of the present invention may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or 35 mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, in a preferred aspect it may be desirable to introduce the pharmaceutical formulations of the invention into the lungs by any suitable route. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. [0166] In a specific aspect, it may be desirable to administer the pharmaceutical formulations of the invention locally 40 to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one aspect, administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue. 45 [0167] In another aspect, the pharmaceutical formulation can be delivered in a biological matrix. An example of a biological matrix is described in U.S. Patent No. 5,962,427, and U.S. Patent Application Publication U.S. 2002/0193338. [0168] In yet another aspect, the pharmaceutical formulation can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another aspect, polymeric materials can 50 be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger & Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351 (1989); Howard et al., 1989, J. Neurosurg. 71:105). In yet another aspect, a controlled release system can be placed in proximity of the composition’s target, i.e., the lung, thus requiring 55 only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533). [0169] The pharmaceutical form ulations of the present invention comprise an effective amount of an attenuated swine

32 EP 2 497 492 B1

influenza virus of the invention, and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical formulation is administered. Saline solutions 5 and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository. Oral formulation can 10 include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Rem- ington’s Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain an effective amount of the therapy, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. The particular formulation may also depend on 15 whether swine influenza virus is live or inactivated. [0170] The amount of the pharmaceutical formulation of the invention which will be effective in the treatment, prevention, management, or amelioration of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of 20 administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient’s circumstances. However, suitable dosage ranges for administration are generally about 102, 5 x 102, 103, 5 x 103, 104, 5 x 104, 105, 5 x 105, 106, 5 x 106, 107, 5 x 107, 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011, 5 x 1011 or 1012 pfu, and most preferably about 10 4 to about 1012 pfu, and can be administered to a subject once, twice, three or more times with intervals as often as needed. Pharmaceutical formulations of the present 25 invention comprising 102, 5 x 102, 103, 5 x 103, 104, 5 x 104, 105, 5 x 105, 106, 5 x 106, 107, 5 x 107, 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 5 x 1011 or 1012 pfu, and most preferably about 104 to about 1012 pfu of an attenuated swine influenza virus of the present invention with altered IFN antagonist activity, can be administered to a subject intranasally, intratracheally, intramuscularly or subcutaneously Effective doses may be extrapolated from dose- response curves derived from in vitro or animal model test systems. 30 [0171] The pharmaceutical formulation of the invention may be used to prevent, manage, treat or ameliorate infections by viruses, bacteria or parasites. The pharmaceutical formulation of the invention may be used to prevent, manage, treat or ameliorate cancer in a pig. The pharmaceutical formulation of the invention may be used to prevent, manage, treat or ameliorate other IFN-treatable diseases.

35 5.7 Therapies Useful in Combination with Swine Influenza Virus

[0172] Disclosed herein are also provides methods for preventing, managing, treating, and/or ameliorating diseases and disorders including, but not limited to, swine influenza virus infections, conditions associated with swine influenza virus infection, infections other than swine influenza virus infections, conditions associated with infections other than 40 swine influenza virus infections, IFN-treatable disorders e.g.,( cancer) and conditions in which an attenuated swine influenza virus of the invention is used as a vector to induce an immune response to an antigen associated with the condition comprising administering to a subject in need thereof an effective amount of one or more attenuated swine influenza viruses disclosed herein and an effective amount of one or more therapies (e.g., prophylactic or therapeutic agents) other than attenuated swine influenza viruses. Therapies include, but are not limited to, small molecules, synthetic 45 drugs, peptides, polypeptides, proteins, nucleic acids e.g.,( DNA and RNA nucleotides including, but not limited to, antisense nucleotide sequences, triple helices, RNAi, and nucleotide sequences encoding biologically active proteins, polypeptides or peptides) antibodies, synthetic or natural inorganic molecules, mimetic agents, and synthetic or natural organic molecules. [0173] Any therapy (e.g., prophylactic or therapeutic agents) which is known to be useful, or which has been used or 50 is currently being used for the prevention, management, treatment, or amelioration of a swine influenza virus infection, infections other than swine influenza virus infections, conditions associated with infections other than swine influenza virus infections, IFN-treatable disorders, conditions in which an attenuated swine influenza virus of the invention is used as a vector to induce an immune response to an antigen associated with the condition, or any other pig disease can be used in combination with an present disclosure attenuated swine influenza virus in accordance with the presentdisclosure. 55 See, e.g., Taylor, Pig Diseases, 6th Ed., Diamond Farm Book Pubns, 1995; Straw et al., Diseases of Swine, 8th Ed., Iowa State University Press, 1999, for information regarding therapies, in particular prophylactic or therapeutic agents, which have been or are currently being used for preventing, treating, managing, and/or ameliorating pig diseases. Examples of prophylactic and therapeutic agents include, but are not limited to, anti-cancer agents; anti-viral agents,

33 EP 2 497 492 B1

anti-inflammatory agents (e.g., adrenocorticoids, corticosteroids (e.g., beclomethasone, budesonide, flunisolide, flutica- sone, triamcinolone, methlyprednisolone, prednisolone, prednisone, hydrocortisone)) and antibiotics ( e.g., dactinomycin (formerly actinomycin), bleomycin, erythomycin, penicillin, mithramycin, and anthramycin (AMC)).

5 5.7.1 Anti-Cancer Therapies

[0174] Any therapy (e.g., therapeutic or prophylactic agent) which is known to be useful, has been used, or is currently being used for the prevention, treatment, management, or amelioration of cancer or one or more symptoms thereof can be used in compositions and methods disclosed herein. Therapies ( e.g., therapeutic or prophylactic agents) include, but 10 are not limited to, peptides, polypeptides, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules. Non-limiting examples of cancer therapies include chem- otherapies, radiation therapies, hormonal therapies, and/or biological therapies/immunotherapies. [0175] In particular aspects, the anti-cancer agent may be, but is not limited to: a chemotherapeutic agent ( e.g., acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, ambomycin; ametantrone acetate, 15 aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, azacitidine, azetepa, batimastat, bleomycin, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, cactinomycin, calusterone, campathecin, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, cyclosporin A, combretastatin A4, combretastatin analogue, cytolytic factor, cytostatin, 20 dacliximab, docetaxel, dacarbazine, dactinomycin, daunorubicin hydrochloride, docetaxel, doxorubicin, droloxifene, dro- mostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, enpromate, epi- propidine, epirubicin hydrochloride, erbulozole, esorubicin hydrochloride, estramustine, etanidazole, etoposide, fazara- bine, fenretinide, floxuridine, fluorouracil, flurocitabine, fosquidone, gemcitabine, hydroxyurea, idarubicin, idarubicin hydrochloride, ifosfamide, ilmofosine, iproplatin, ifosfamide, irinotecan hydrochloride, lanreotide acetate, letrozole, leupro- 25 lide acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, mercap- topurine, methotrexate, metoprine, meturedepa, mitindomide, mitocarcin, mitocromin, mitomycin, mitoxantrone, myco- phenolic acid, nitrosoureas, nocodazole, nogalamycin, ormaplatin, oxisuran, paclitaxel, plicamycin, platinum complex, platinum compounds, platinum-triamine complex, procarbizine, puromycin, taxol, thioguanine, talisomycin, tecogalan sodium, tegafur, teloxantrone hydrochloride, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate , vindesine, 30 vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, and zinostatin), matrix metalloproteinase inhibitors, tyrosine kinase inhibitors, tyrphostins, urokinase receptor antagonists, multiple drug resistance gene inhibitor, multiple tumor suppressor 1-based therapy, plasminogen activator inhibitor, bisphosphonates (e.g., pamidronate (Aredria), sodium clondronate (Bonefos), zoledronic acid (Zometa), alendronate (Fosamax), etidronate, ibandornate, cimadronate, risedromate, and tiludromate), 35 a cytokine (e.g., IL-2, IFN-α, IFN-β, IFN-γ, leukemia inhibiting factor, leukocyte alpha interferon, human chorionic gona- dotrophin, and thrombopoietin), a hormone (e.g., thyroid stimulating hormone), an antibody (e.g., an anti-CD2 antibody, an anti-CD20 antibody, and an antibody immunospecific for one or more galanin receptors), vitamin D (e.g., 20-epi-1,25 dihydroxyvitaminD3), an angiogenesis inhibitor, an antisense oligonucleotide,an apoptosis genemodulator, an apoptosis regulator, a BCR/ABL antagonist, a cartilage derived inhibitor, an estrogen agonist, an estrogen antagonist, a gelatinase 40 inhibitor, a glutathione inhibitor, an HMG CoA reductase inhibitor e.g( ., atorvastatin, cerivastatin, fluvastatin, lescol, lupitor, lovastatin, rosuvastatin, and simvastatin), an immunostimulant peptide, an insulin-like growth factor-1 receptor inhibitor, an interferon agonist, leuprolide+estrogen+progesterone, leuprorelin, a mismatched double stranded RNA, a proteasome inhibitor, a protein A-based immune modulator, a protein kinase C inhibitor, a protein tyrosine phosphatase inhibitor, a raf antagonist, a ras farnesyl protein transferase inhibitor, a ribozyme, RNAi, a signal transduction modulator, 45 a stem celi inhibitor, a stem-cell division inhibitor, a telomerase inhibitor, a thymopoietin receptor agonist, and a translati on inhibitor. [0176] In specific aspects, radiation therapy comprising the use of x-rays, gamma rays and other sources of radiation to destroy the cancer cells is used in combination with the attenuated swine influenza viruses disclosed herein. In preferred aspects, the radiation treatment is administered as external beam radiation or teletherapy, wherein the radiation 50 is directed from a remote source. In other preferred aspects, the radiation treatment is administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass. [0177] Cancer therapies and their dosages, routes of administration and recommended usage are known in the art and have been described in such literature as the Physicians’ Desk Reference (58th ed., 2004) and Merck Veterinary Manual (8th ed., 1998). 55 5.7.2 Anti-angiogenesis Therapies

[0178] Any anti-angiogenic agent well-known to one of skill in the art can be used in the compositions and methods

34 EP 2 497 492 B1

disclosed herein. Non-limiting examples of anti-angiogenic agents include proteins, polypeptides, peptides, fusion proteins, antibodies (e.g., chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab fragments, F(ab)2 fragments, and antigen- binding fragments thereof) such as antibodies that immunospecifically bind to TNFα, nucleic acid molecules (e.g., antisense molecules or triple helices), organic molecules, inorganic molecules, and small molecules that reduce or inhibit 5 or neutralizes the angiogenesis. In particular, examples of anti-angiogenic agents, include, but are not limited to, en- dostatin, angiostatin, apomigren, anti-angiogenic antithrombin III, the 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragments of fibronectin, a uPA receptor antagonist, the 16 kDa proteolytic fragment of prolactin, the 7.8 kDa proteolyticfragment of platelet factor-4, the anti-angiogenic24 amino acid fragment of platelet factor-4, the anti-angiogenic factor designated 13.40, the anti-angiogenic 22 amino acid peptide fragment of thrombospondin I, the anti-angiogenic 10 20 amino acid peptide fragment of SPARC, RGD and NGR containing peptides, the small anti-angiogenic peptides of

laminin, fibronectin, procollagen and EGF, integrin αvβ3 antagonists (e.g., anti-integrin αvβ3 antibodies), acid fibroblast growth factor (aFGF) antagonists, basic fibroblast growth factor (bFGF) antagonists, vascular endothelial growth factor (VEGF) antagonists (e.g., anti-VEGF antibodies), and VEGF receptor (VEGFR) antagonists ( e.g., anti-VEGFR antibodies). 15 5.7.3 Anti-viral Therapies

[0179] Any anti-viral agent well-known to one of skill in the art can be used in the compositions and the methods disclosed herein. Non-limiting examples of anti-viral agents include proteins, polypeptides, peptides, fusion proteins 20 antibodies, nucleic acid molecules, organic molecules, inorganic molecules, and small molecules that inhibit and/or reduce the attachment of a virus to its receptor, the internalization of a virus into a cell, the replication of a virus, or release of virus from a cell. In particular, anti-viral agents include, but are not limited to, nucleoside analogs(e.g., zidovudine, acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, and ribavirin), foscarnet, amantadine, rimanta- dine, saquinavir, indinavir, ritonavir, alpha-interferons and other interferons, and AZT. 25 [0180] In specific aspects, the anti-viral agent is an immunomodulatory agent that is immunospecific for a viral antigen. As used herein, the term "viral antigen" includes, but is not limited to, any viral peptide, polypeptide and protein e.g( ., 3AB, 3ABC of foot-and-mouth disease virus, GP5 of porcine reproductive and respiratory syndrome virus, pseudorabies virus gE, swine transmissible gastroenteritis virus nucleoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, herpes simplex virus glycoprotein (e.g., gB, gC, gD, and gE) and hepatitis B surface antigen) that is capable of 30 eliciting an immune response. Antibodies useful in this aspect for treatment of a viral infectious disease include, but are not limited to, antibodies againstantigens of pathogenic viruses, including as examples and not by limitation: adenovirdiae (e.g., mastadenovirus and aviadenovirus), herpesviridae (e.g., herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 5, and herpes simplex virus 6), leviviridae (e.g., levivirus, enterobacteria phase MS2, allolevirus), poxviridae (e.g., chordopoxvirinae, parapoxvirus, avipoxvirus, capripoxvirus, leporiipoxvirus, suipoxvirus, molluscipoxvirus, and 35 entomopoxvirinae), papovaviridae e.g ( ., polyomavirus and papillomavirus), paramyxoviridaee.g ( ., paramyxovirus, parainfluenza virus 1, mobillivirus (e.g., measles virus), rubulavirus (e.g., mumps virus), pneumonovirinae (e.g., pneumovirus, human respiratory synctial virus), and metapneumovirus (e.g., avian pneumovirus and human metapneumovirus)), picornaviridae (e.g., enterovirus, rhinovirus, hepatovirus (e.g., human hepatits A virus), cardiovirus, and aptho- virus), reoviridae (e.g., orthoreovirus, orbivirus, rotavirus, cypovirus, fijivirus, phytoreovirus, and oryzavirus), retroviridae 40 (e.g., mammalian type B retroviruses, mammalian type C retroviruses, avian type C retroviruses, type D retrovirus group, BLV-HTLV retroviruses, lentivirus (e.g., human immunodeficiency virus 1 and human immunodeficiency virus 2), spu- mavirus), flaviviridae (e.g., hepatitis C virus), hepadnaviridae ( e.g., hepatitis B virus), togaviridae ( e.g., alphavirus (e.g., sindbis virus) and rubivirus (e.g., rubella virus)), rhabdoviridae ( e.g., vesiculovirus, lyssavirus, ephemerovirus, cytorhab- dovirus, and necleorhabdovirus), arenaviridae ( e.g., arenavirus, lymphocytic choriomeningitis virus, Ippy virus, and lassa 45 virus), and coronaviridae (e.g., coronavirus and torovirus). [0181] Specific examples of antibodies available useful for the treatment of a viral infectious disease include, but are not limited to, PRO542 (Progenics) which is a CD4 fusion antibody useful for the treatment of HIV infection; Ostavir (Protein Design Labs, Inc., CA) which is a human antibody useful for the treatment of hepatitis B virus; and Protovir (Protein Design Labs, Inc., CA) which is a humanized lgG1 antibody useful for the treatment of cytomegalovirus (CMV). 50 [0182] Anti-viral therapies and their dosages, routes of administration and recommended usage are known in the art and have been described in such literature as the Physicians’ Desk Reference (58th ed., 2004) and Merck.Veterinary Manual(8thed., 1998). Additional information on viral infections is available in Cecil Textbookof Medicine (18th ed., 1988).

5.7.4 Antibiotic Therapies 55 [0183] Any antibiotic agent well-known to one of skill in the art can be used in the compositions and the methods disclosed herein. Antibacterial agents or antibiotics that can be used in combination with the complexes disclosed herein include but are not limited to: aminoglycoside antibiotics ( e.g., apramycin, arbekacin, bambermycins, butirosin, dibekacin,

35 EP 2 497 492 B1

neomycin, neomycin, undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, and spectinomycin), amphenicol antibiotics (e.g., azidamfenicol, chloramphenicol, florfenicol, and thiamphenicol), ansamycin antibiotics e.g( ., rifamide and rifampin), carbacephems (e.g., loracarbef), carbapenems (e.g., biapenem and imipenem), cephalosporins e.g( ., cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, and cefpirome), ce- 5 phamycins (e.g., cefbuperazone, cefmetazole, and cefminox), monobactamse.g (., aztreonam, carumonam, and tigemonam), oxacephems (e.g., flomoxef, and moxalactam), penicillins (e.g., amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium, epicillin, fenbenicillin, floxacillin, penamccillin, penethamate hydriodide, penicillin o-benethamine, penicillin 0, penicillin V, penicillin V benzathine, penicillin V hydrabamine, penimepicycline, and phencihicillin potassium), lincosamides e.g( ., clindamycin, and lincomycin), macrolides 10 (e.g., azithromycin, carbomycin, clarithomycin, dirithromycin, erythromycin, and erythromycin acistrate), amphomycin, bacitracin, capreomycin, colistin, enduracidin, enviomycin, tetracyclines ( e.g., apicycline, chlortetracycline, clomocycline, anddemeclocycline), 2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans ( e.g., furaltadone, and furazolium chloride), quinolones and analogs thereof (e.g., cinoxacin, ciprofloxacin, clinafloxacin, flumequine, and grepagloxacin), sulfona- mides (e.g., acetyl sulfamethoxypyrazine, benzylsulfamide, noprylsulfamide, phthalylsulfacetamide, sulfachrysoidine, 15 and sulfacytine), sulfones (e.g., diathymosulfone, glucosulfone sodium, and solasulfone), cycloserine, mupirocin and tuberin. [0184] Additional examples of antibacterial agents include but are not limited to Acedapsone; Acetosulfone Sodium; Alamecin; Alexidine; Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin; Amifloxacin Mesylate; Amikacin; Ami- kacin Sulfate; Aminosalicylic acid; Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; Ampicillin Sodium; 20 Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate; Avilamycin; Avoparcin; Azithromycin; Azlocillin; Azlocillin Sodium; Bacampicillin Hydrochloride; Bacitracin; Bacitracin Methylene Disalicylate; Bacitracin Zinc; Bambermycins; Benzoylpas Calcium; Berythromycin; Betamicin Sulfate; Biapenem; Biniramycin; Biphenamine Hydrochloride; Bispy- rithione Magsulfex; Butikacin; Butirosin Sulfate; Capreomycin Sulfate; Carbadox; Carbenicillin Disodium; Carbenicillin Indanyl Sodium; Carbenicillin Phenyl Sodium; Carbenicillin Potassium; Carumonam Sodium; Cefaclor; Cefadroxil; Ce- 25 famandole; Cefamandole Nafate; Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium; Cefazolin; Cefazo- lin Sodium; Cefbuperazone; Cefdinir; Cefepime; Cefepime Hydrochloride; Cefetecol; Cefixime; Cefmnenoxime Hydro- chloride; Cefmetazole; Cefmetazole Sodium; Cefonicid Monosodium; Cefonicid Sodium; Cefoperazone Sodium; Ce- foranide; Cefotaxime Sodium; Cefotetan; Cefotetan Disodium; Cefotiam Hydrochloride; Cefoxitin; Cefoxitin Sodium; Cefpimizole; Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium; Cefpirome Sulfate; Cefpodoxime Proxetil; Cefpro- 30 zil; Cefroxadine; Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium; Ceftriaxone Sodium; Cefuroxime; Cefuroxime Axetil; Cefuroxime Pivoxetil; Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; Cephalexin Hydrochlo- ride; Cephaloglycin; Cephaloridine; Cephalothin Sodium; Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride; Cetophenicol;Chloramphenicol; Chloramphenicol Palmitate; ChloramphenicolPantothenate Complex; Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilate; Chloroxylenol; Chlortetracycline Bisulfate; Chlortetracycline Hydro- 35 chloride; Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride; Cirolemycin; Clarithromycin; Clinafloxacin Hydrochlo- ride; Clindamycin; Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride; Clindamycin Phosphate; Clofaz- imine; Cloxacillin Benzathine; Cloxacillin Sodium; Cloxyquin; Colistimethate Sodium; Colistin Sulfate; Coumermycin; Coumermycin Sodium; Cyclacillin; Cycloserine; Dalfopristin; Dapsone; Daptomycin; Demeclocycline; Demeclocycline Hydrochloride; Demecycline; Denofungin; Diaveridine; Dicloxacillin; Dicloxacillin Sodium; Dihydrostreptomycin Sulfate; 40 Dipyrithione; Dirithromycin; Doxycycline; Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline Hyclate; Droxacin Sodium; Enoxacin; Epicillin; Epitetracycline Hydrochloride; Erythromycin; Erythromycin Acistrate; Erythromycin Estolate; Erythromycin Ethylsuccinate; Erythromycin Gluceptate; Erythromycin Lactobionate; Erythromycin Propionate; Erythro- mycin Stearate; Ethambutol Hydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine; Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin; Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic Acid; 45 Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin; Hetacillin; Hetacillin Potassium; Hexedine; Ibafloxacin; Imi- penem; Isoconazole; Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin; Levofuraltadone; Levopropyl- cillin Potassium; Lexithromycin; Lincomycin; Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin Hydrochloride; Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline; Meclocycline Sulfosalicylate; Megalomicin Potassium Phosphate; Mequidox; Meropenem; Methacycline; Methacycline Hydrochloride; Methenamine; Methenamine Hippurate; 50 Methenamine Mandelate; Methicillin Sodium; Metioprim; Metronidazole Hydrochloride; Metronidazole Phosphate; Me- zlocillin; Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; Mirincamycin Hydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium; Nalidixate Sodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin Palmitate; Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate; Neutramycin; Nifuradene; Nifuraldezone; Nifuratel; Nifuratrone; Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol; Nifurthiazole; Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novo- 55 biocin Sodium; Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium; OxolinicAcid; Oxytetracycline; Oxytetracycline Calcium; Oxytetracycline Hydrochloride; Paldimycin; Parachlorophenol; Paulomycin; Pefloxacin; Pe- floxacin Mesylate; Periamecillin; Penicillin G Benzathine; Penicillin G Potassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V; Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin V Potassium; Pentizidone Sodium;

36 EP 2 497 492 B1

Phenyl Aminosalicylate; Piperacillin Sodium; Pirbenicillin Sodium; Piridicillin Sodium; Pirlimycin Hydrochloride; Pivamp- icillin Hydrochloride; Pivampicillin Pamoate; Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin; Propikacin; Pyrazinamide; Pyrithione Zinc; QuindecamineAcetate; Quinupristin; Racephenicol; Ramoplanin; Ranimycin; Relomycin; Repromicin; Rifabutin; Rifametane; Rifamexil; Rifamide; Rifampin; Rifapentine; Rifaximin; Rolitetracycline; Rolitetracy- 5 cline Nitrate; Rosaramicin; Rosaramicin Butyrate; Rosaramicin Propionate; Rosaramicin Sodium Phosphate; Rosara- micin Stearate; Rosoxacin; Roxarsone; Roxithromycin; Sancycline; Sanfetrinem Sodium; Sarmoxic illin; Sarpicillin; Sco- pafingin; Sisomicin; Sisomicin Sulfate; Sparfloxacin; Spectinomycin Hydrochloride; Spiramycin; Stallimycin Hydrochlo- ride; Steffimycin; Streptomycin Sulfate; Streptonicozid; Sulfabenz; Sulfabenzamide; Sulfacetamide; Sulfacetamide So- dium; Sulfacytine; Sulfadiazine; Sulfadiazine Sodium; Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfameth- 10 azine; Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine; Sulfamoxole; Sulfanilate Zinc; Sulfanitran; Sulfasala- zine; Sulfasomizole; Sulfathiazole; Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl; Sulfisoxazole Diolamine;Sulfomyxin; Sulopenem; Sultamicillin; Suncillin Sodium; Talampicillin Hydrochloride; Teicoplanin; Temafloxacin Hydrochloride; Te- mocillin; Tetracycline; Tetracycline Hydrochloride; Tetracycline Phosphate Complex; Tetroxoprim; Thiamphenicol; Thiphencillin Potassium; Ticarcillin Cresyl Sodium; Ticarcillin Disodium; Ticarcillin Monosodium; Ticlatone; Tiodonium 15 Chloride; Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; Trimethoprim Sulfate; Trisulfapyrimidines; Trole- andomycin; Trospectomycin Sulfate; Tyrothricin; Vancomycin; Vancomycin Hydrochloride; Virginiamycin; Zorbamycin. [0185] Antibiotic therapies and their dosages, routes of administration and recommended usage are known in the art and have been described in such literature as the Physicians’ Desk Reference (58th ed., 2004) and Merck Veterinary Manual (8th ed:, 1998). 20 5.7.5 Immunomodulatory Therapies

[0186] Any immunomodulatory agent well-known to one of skill in the art may be used in the methods and compositions disclosed herein. Immunomodulatory agents can affect one or more or all aspects of the immune response in a subject. 25 Aspects of the immune response include, but are not limited to, the inflammatory response, the complement cascade, leukocyte and lymphocyte differentiation, proliferation, and/or effector function, monocyte and/or basophil counts, and the cellular communication among cells of the immune system. In certain aspects disclosed herein, an immunomodulatory agent modulates one aspect of the immune response. In other aspects, an immunomodulatory agent modulates more than one aspect of the immune response. In a preferred aspect, the administration of an immunomodulatory agent to a 30 subject inhibits or reduces one or more aspects of the subject’s immune response capabilities. In certain aspects, an immunomodulatory agent is not an anti-inflammatory agent. In certain aspects, an immunomodulatory agent is not an anti-angiogneic agent. In certain aspects, an immunomodulatory agent is a chemotherapeutic agent. In certain aspects, an immunomodulatory agent is not a chemotherapeutic agent. [0187] Examples of immunomodulatory agents include, but are not limited to, proteinaceous agents such as cytokines, 35 peptide mimetics, and antibodies ( e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)2 fragmentsor epitope binding fragments),nucleic acid molecules ( e.g.,antisense nucleic acid molecules and triple helices), small molecules, organic compounds, and inorganic compounds. In particular, immunomodulatory agents include, but are not limited to, methotrexate, leflunomide, cyclophosphamide, cytoxan, Immune, cyclosporine A, minocycline, aza- thioprine, antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, steroids, mycophenolate 40 mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), T cell receptor modulators, and cytokine receptor modulators. [0188] . Examples of T cell receptor modulators include, but are not limited to, anti-T cell receptor antibodies e.g( ., anti-CD4 antibodies e.g ( ., cM-T412 (Boeringer), IDEC-CE9.1® (IDEC and SKB), mAB 4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD2 antibodies, anti-CD3 antibodies ( e.g., Nuvion (Product Design Labs), OKT3 (John- 45 son & Johnson), or Rituxan (IDEC)), anti-CD5 antibodies e.g( ., an anti-CD5 ricin-linked immunoconjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies e.g( ., IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH 1H (Ilex)), anti-CD2 antibodies, anti-CD11a antibodies (e.g., Xanelim (Genentech)), and anti-B7 antibodies (e.g., IDEC-114) (IDEC))), CTLA4-immunoglobulin, and LFA-3TIP (Biogen, Inter- national Publication No. WO 93/08656 and U.S. Patent No. 6,162,432). 50 [0189] Examples of cytokine receptor modulators include, but are not limited to, soluble cytokine receptors (e.g., the extracellular domain of a TNF- α receptor or a fragment thereof, the extracellular domain of an IL-1 β receptor or a fragment thereof, and the extracellular domain of an IL-6 receptor or a fragment thereof), cytokines or fragments thereof e.g( ., interleukin IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-23, TNF-α, TNF-β, interferon (IFN)-α, IFN-β, IFN-γ, and GM-CSF), anti-cytokine receptor antibodies (e.g., anti-IFN receptor antibodies, anti-IL-2 re- 55 ceptor antibodies, (e.g., Zenapax (Protein Design Labs)), anti-IL-3 receptor antibodies, anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies, anti-IL-9 receptor antibodies anti-IL-10 receptor antibodies, anti-IL-12 receptor antibodies, anti-IL-13 receptor antibodies, anti-IL-15 receptor antibodies, and anti-IL-23 receptor antibodies), anti-cytokine antibodies (e.g., anti-IFN antibodies, anti-TNF-α antibodies, anti-IL-1β antibodies, anti-IL-3 antibodies, anti-IL-6 antibodies, anti-IL-

37 EP 2 497 492 B1

8 antibodies (e.g., ABX-IL-8 (Abgenix)), anti-IL-9 antibodies anti-IL-12 antibodies, anti-IL-13 antibodies, anti-IL-15 antibodies, and anti-IL-23 antibodies). [0190] In a specific aspect, a cytokine receptor modulator is IL-3, IL-4, IL-10, or a fragment thereof. In another aspect, a cytokine receptor modulator is an anti-IL-1β antibody, anti-IL-6 antibody, anti-IL-12 receptor antibody, or anti-TNF-α 5 antibody. In another aspect a cytokine receptor modulator is the extracellular domain of a TNF- α receptor or a fragment thereof. [0191] In certain aspects, an immunomodulatory agent is a B cell receptor modulator. Examples of B cell receptor modulators include, but are not limited to, CD 19. Targeting B cell receptors provides one means for modulating B cells. [0192] In a preferred aspect, proteins, polypeptides or peptides (including antibodies) that are utilized as immunomod- 10 ulatory agents are derived from the same species as the recipient of the proteins, polypeptides or peptides so as to reduce the likelihood of an immune response to those proteins, polypeptides or peptides. In another preferred aspect, when the subject is a pig, the proteins, polypeptides, or peptides that are utilized as immunomodulatory agents are derived from pig. [0193] In accordance with the aspects disclosed herein, one or more immunomodulatory agents are administered to 15 a subject prior to, subsequent to, or concomitantly with an attenuated swine influenza virus. Any technique well-known to one skilled in the art can be used to measure one or more aspects of the immune response in a particular subject, and thereby determine when it is necessary to administer an immunomodulatory agent to said subject. In a preferred aspect, a mean absolute lymphocyte count of approximately 500 cells/mm3, preferably 600 cells/mm3, 650 cells/mm3, 700 cells/mm3, 750 cells/mm3, 800 cells/mm3, 900 cells/mm3, 1000 cells/mm3, 1100 cells/mm3, or 1200 cells/mm3 is 20 maintained in a subject [0194] Preferably, agents that are commercially available and known to function as immunomodulatory agents are used in accordance with the methods disclosed herein. The immunomodulatory activity of an agent can be determined in vitro and/or in vivo by any technique well-known to one skilled in the art, including, e.g., by CTL assays, proliferation assays, and immunoassays (e.g. ELISAs) for the expression of particular proteins such as co-stimulatory molecules 25 and cytokines.

5.8 Dosage & Frequency of Administration

[0195] The amount of a prophylactic or therapeutic agent or a composition disclosed herein which will be effective in 30 the prevention, treatment, management, and/or amelioration of a swine influenza virus infection or a condition associated therewith, an infection other than a swine influenza virus infection or a condition associated therewith, an IFN-treatable disease or a condition in which an attenuated swine influenza virus is used as a vector to induce an immune response to an antigen associated with the condition, or the prevention of the recurrence, onset, or development of one or more symptoms of a swine influenza virus infection or a condition associated therewith, an infection other than a swine influenza 35 virus infection or a condition associated therewith, an IFN-treatable disease, or a condition in which an attenuated swine influenza virus is used as a vector to induce an immune response to an antigen associated with the condition, can be determined by standard clinical methods. The frequency and dosage will vary,also according to factors specific for each patient depending on the specific therapies ( e.g., the specific therapeutic or prophylactic agent or agents) administered, the severity of the disorder, disease, or condition, the route of administration, as well as age, body, weight, response, 40 and the past medical history of the patient. For example, the dosage of a prophylacticor therapeutic agent or a composition which will be effective in the treatment, prevention, management, and/or amelioration of a swine influenza virus infection or a condition associated therewith, a viral infection other than a swine influenza virus infection or a condition associated therewith, or an IFN-treatable disease, or the prevention of the recurrence, onset, or development of one or more symptoms of a swine influenza virus infection or a condition associated therewith, a viral infection other than a swine 45 influenza virus infection or a condition associated therewith, or an IFN-treatable disease, can be determined by animal models such as those known to those skilled in the art. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages as reported in literature and recommended in the Physician’s Desk Reference (58th ed., 2004), Merck Veterinary Manual (8th ed., 1998), or Straw et al., Diseases of the Swine, Iowa State University 50 Press (1999). [0196] Exemplary doses of a vaccine or immunogenic formulation include 102 to about 108, about 103 to about 107, about 104 to about 5 x 10 6 pfu or 10 4 to about 10 7 pfu. In other aspects, the dose of a vaccine or immunogenic formulation disclosed herein administered to a subject is 10 2, 5 x 102, 103, 5 x 103, 104, 5 x 104, 105, 5 x 105, 106, 5 x 106, 107, 5 x 107 or 108 pfu. Exemplary doses of a pharmaceutical formulation include 102, 5 x 102, 103, 5 x 103, 104, 5 x 104, 105, 55 5 x 105, 106, 5 x 106, 107, 5 x 107, 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011, 5 x 1011 or 1012 pfu. In certain aspects, the dose of the pharmaceutical formulation administered to the subject is between about 102 to about 1012, about 102 to about 1010, about 102 to about 108, about 103 to about 109, about 103 to about 107, about 104 to about 108, about 104 to about 5 x 106 pfu or about 104 to about 1012 pfu.

38 EP 2 497 492 B1

[0197] Exemplary doses of a small molecule include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight e.g( ., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligram’s per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). 5 [0198] For antibodies, proteins, polypeptides, peptides and fusion proteins disclosed herein, the dosage administered to a patient is typically 0.0001 mg/kg to 100 mg/kg of the patient’s body weight. Preferably, the dosage administered to a patient is between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient’s 10 body weight. Generally, pig antibodies have a longer half-life within the pig than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of pig antibodies and less frequent administration to pigs is often possible. Further, the dosage and frequency of administration of antibodies or fragments thereof may be reduced by enhancing uptake and tissue penetration of the antibodies by modifications such as, for example, lipidation. [0199] Preferably, the dosages of prophylactic or therapeutic agents used in combination therapies disclosed herein 15 are lower than those which have been or are currently being used to prevent, treat, manage, and/or ameliorate a swine influenza virus infection or a condition or symptoms associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease, or a condition in which an attenuated swine influenza virus is used as a vector to induce an immune response to an antigen associated with the condition. The recommended dosages of agents currently used for the prevention, treatment, management, or amelioration of a 20 swine influenza virus infection or a condition or symptoms associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease, or a condition in which an attenuated swine influenza virus is used as a vector to induce an immune response to an antigen associated with the condition, can be obtained from any reference in the art including, but not limited to, Hardman et al., eds., 2001, Goodman & Gilman’s The Pharmacological Basis Of Basis Of Therapeutics, 10th ed., Mc-Graw-Hill, New York; Physicians’ Desk 25 Reference (PDR) 58th ed., 2004, Medical Economics Co., Inc., Montvale, NJ, and Merck Veterinary Manual (8th ed., 1998); Taylor, Pig Diseases, 6th Ed., Diamond Farm Book Pubns, 1995; and Straw et al., Diseases of Swine, 8th Ed., Iowa State University Press, 1999. [0200] In various aspects, the therapies ( e.g., prophylactic or therapeutic agents) are administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours 30 to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 35 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours part. In preferred aspects, two or more therapies are administered within the same patent visit. [0201] In certain aspects, oneor more compositions disclosed hereinand one or more other therapies( e.g.,prophylactic or therapeutic agents) are cyclically administered. Cycling therapy involves the administration of a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (e.g., a 40 second prophylactic or therapeutic agent) for a period of time, optionally, followed by the administration of a third therapy (e.g., prophylactic or therapeutic agent) for a period of time and so forth, and repeating this sequential administration, i.e., the cycle in order to reduce the development of resistance to one of the therapies, to avoid or reduce the side effects of one of the therapies, and/or to improve the efficacy of the therapies. [0202] A vaccine or immunogenic composition of the invention may be administered to a subject at a single dose 45 followed by a second dose 3 to 6 weeks later. Booster vaccinations may be administered to the subject at 6 to 12 month intervals following the second vaccination. The subject may be mammal. The subject may be a pig with a swine influenza A infection. [0203] The administration of the same compostions of the invention may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, 50 or at least 6 months. The administration of the same therapye.g ( ., prophylactic or therapeutic agent) other than a composition of the invention may be repeated and the administration may be separated by at least at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.

5.9 Biological Assays 55 5.9.1 In vitro assays

[0204] Growth of the attenuated swine influenza viruses of the present invention can be assessed in pig cells, e.g.,

39 EP 2 497 492 B1

PK cells, other pig cells as described herein, and other IFN-competent and IFN-deficient cells. In a specific aspect, PK cells are infected at a MOI of 0.0005 and 0.001, 0.001 and 0.01, 0.01 and 0.1, 0.1 and 1, or 1 and 10, or a MOI of 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 or 10 and incubated with serum free media supplemented with 5% allantoic fluid (F. Cook, University of Kentucky, Lexington, KY). Viral titers are determined in the supernatant by HA plaqued in MDCK 5 cells as described below. Other cells in which viral titers can be assessed include, but are not limited to, PK cells, Vero cells, primary human umbilical vein endothelial cells (HUVEC), H292 human epithelial cell line, HeLa cells, and swine embryonic kidney cells RES. [0205] Viral assays include those that measure altered viral replication (as determined, e.g., by plaque formation) or the production of viral proteins (as determined, e.g., by western blot analysis) or viral RNAs (as determined, e.g., by RT- 10 PCR or northern blot analysis) in cultured cells in vitro using methods which are well known in the art. [0206] Induction of IFN responses may be determined by measuring the phosphorylated state of components of the IFN pathway following infection with the test mutant virus,e .g., IRF-3, which is phosphorylated in response to dou- blestranded RNA. In response to type I IFN, Jak1 kinase and TyK2 kinase, subunits of the IFN receptor, STAT1, and STAT2 are rapidly tyrosine phosphorylated. Thus, in order to determine whether the attenuated swine influenza virus 15 induces IFN responses, cells, such as 293 cells, are infected with the test mutant virus and following infection, the cells are lysed. IFN pathway components, such as Jak1 kinase or TyK2 kinase, are immunoprecipitated from the infected cell lysates, using specific polyclonal sera or antibodies, and the tyrosine phosphorylated state of the kinase is determined by immunoblot assays with an anti-phosphotyrosine antibody(e.g., see Krishnan et al. 1997, Eur. J. Biochem. 247:298-305). An enhanced phosphorylated state of any of the components of the IFN pathway following infection with 20 the attenuated swine influenza virus would indicate induction of IFN responses by the attenuated swine influenza virus. [0207] Induction of IFN responses may be determined by measuring IFN-dependent transcriptional activation following infection with the test attenuated swine influenza virus. In this embodiment, the expression of genes known to be induced by IFN, e.g., Mx, PKR, 2-5- oligoadenylatesynthetase, major histocompatibility complex (MHC) class I, etc., can be analyzed by techniques known to those of skill in the art (e.g., northern blots, western blots, PCR, etc.). Alternatively, 25 test cells such as embryonic kidney cells or osteogenic sarcoma cells, are engineered to transiently or constitutively express reporter genes such as luciferase reporter gene or chloramphenicol transferase (CAT) reporter gene under the control of an interferon stimulated response element, such as the IFN-stimulated promoter of the ISG-54K gene (Bluyssen et al., 1994, Eur. J. Biochem. 220:395-402). Cells are infected with the test attenuated swine influenza virus and the level of expression of the reporter gene compared to that in uninfected cells or cells infected with wild-type virus. An 30 increase in the level of expression of the reporter gene following infection with the test virus would indicate that the test attenuated swine influenza virus is inducing an IFN response. [0208] Measuring IFN induction can also be assessed by determining whether an extract from the cell or egg infected with the test attenuated swine influenza virus is capable of conferring protective activity against viral infection. More specifically, groups of 10-12 day old embryonated chicken eggs or 10-day old embryonated chicken eggs are infected 35 with the test attenuated swine influenza virus or the wild-type virus. Approximately 15 to 20 hours post infection, the allantoic fluid is harvested and tested for IFN activity by determining the highest dilution with protective activity against swine influenza virus infection in tissue culture cells; such as MDCK cells.

5.9.2 In vivo assays 40 [0209] Decreased virulence of the attenuated swine influenza viruses of the present invention can be assessed in a subject, in particular, pigs. In one example, the ability to induce lung lesions and cause infection in swine is compared to wild-type swine influenza virus and mock virus. Lung lesions can be assessed as a percentage of lung lobes that are healthy by visual inspection. Animals are euthanized 5 days p.i. by intravenous administration of pentobarbital, and their 45 lungs are removed in toto. The percentage of the surface of each pulmonary lobe that is affected by macroscopic lesions is estimated visually. The percentages are averaged to obtain a mean value for the 7 pulmonary lobes of each animal. [0210] In other assays, nasal swabs and BALF can be tested to determine virus burden or titer. Nasal swabs can be taken during necropsy to determine viral burden post-infection. Samples can also be taken with care from live pigs to determine viral burden post-infection. BALF can be obtained after the lungs are removed from the thoracic cavity by 50 rinsing the lungs (via trachea) with about 30 ml of McCoy’s medium without serum. [0211] For quantitation of virus in tissue samples, tissue samples are homogenized in phosphate-buffered saline (PBS), and dilutions of clarified homogenates adsorbed for 1 h at 37°C onto monolayers of MDCK cells. Infected monolayers are then overlaid with a solution of minimal essential medium containing 0.1% bovine serum albumin (BSA),

0.01% DEAE-dextran, 0.1% NaHCO3, and 1% agar. Plates are incubated 2 to 3 days until plaques could be visualized. 55 Tissue culture infectious dose (TCID) assays to titrate virus from PR8-infected samples are carried out as follows. Confluent monolayers of MDCK cells in 96-well plates are incubated with log dilutions of clarified tissue homogenates in media. Two to three days after inoculation, 0.05-ml aliquots from each well are assessed for viral growth by hemagglutination assay (HA assay).

40 EP 2 497 492 B1

[0212] In yet other assays, histopathologic evaluations are performed after infection. The nasal turbinates and trachea are examined for epithelial changes and subepithelial inflammation. The lungs are examined for bronchiolar epithelial changes and peribronchiolar inflammation in large, medium, and small or terminal bronchioles. The alveoli are also evaluated for inflammatory changes. The medium bronchioles are graded on a scale of 0 to 3+ as follows: 0 (normal: 5 lined by medium to tall columnar epithelial cells with ciliated apical borders and basal pseudostratified nuclei; minimal inflammation); 1+ (epithelial layer columnar and even in outline with only slightly increased proliferation; cilia still visibl e on many cells); 2+ (prominent changes in the epithelial layer ranging from attenuation to marked proliferation; cells disorganized and layer outline irregular at the luminal border); 3+ (epithelial layer markedly disrupted and disorganized with necrotic cells visible in the lumen; some bronchioles attenuated and others in marked reactive proliferation). 10 [0213] The trachea is graded on a scale of 0 to 2.5+ as follows: 0 (normal: Lined by medium to tall columnar epithelial cells with ciliated apical border, nuclei basal and pseudostratified. Cytoplasm evident between apical border and nucleus. Occasional small focus with squamous cells); 1+ (focal squamous metaplasia of the epithelial layer); 2+ (diffuse squamous metaplasia of much of the epithelial layer, cilia may be evident focally); 2.5+ (diffuse squamous metaplasia with very few cilia evident). 15 [0214] Swine influenza virus immunohistochemistry is performed using a NP-specific monoclonal antibody. Staining is graded 0 to 3+ as follows: 0 (no infected cells); 0.5+ (few infected cells); 1+ (few infected cells, as widely separated individual cells); 1.5+ (few infected cells, as widely separated singles and in small clusters); 2+ (moderate numbers of infected cells, usually affecting clusters of adjacent cells in portions of the epithelial layer lining bronchioles, or in smal l sublobular foci in alveoli); 3+ (numerous infected cells, affecting most of the epithelial layer in bronchioles, or widespread 20 in large sublobular foci in alveoli).

5.9.3 Determining Viral Titer

[0215] Viral titer is determined by inoculating serial dilutions of swine influenza virus into cell cultures ( e.g., pig cultures), 25 chick embryos, or live animals (e.g., pigs). After incubation of the virus for a specified time, the virus is isolated using standard methods. [0216] Physical quantitation of virus titer can be performed using PCR applied to viral supernatants (Quinn & Trevor, 1997; Morgan et al., 1990), hemagglutination assays, tissue culture infectious doses (TCID50) or egg infectious doses (EID50). 30 [0217] The HA assay is carried out in V-bottom 96-well plates. Serial twofold dilutions of each sample in PBS were incubated for 1 h on ice with an equal volume of a 0.5% suspension of chicken erythrocytes in PBS. Positive wells contained an adherent, homogeneous layer of erythrocytes; negative wells contain a nonadherent pellet.

5.9.4 Determining Hemagglutination Inhibiting Antibody Titers 35 [0218] In one method, HA inhibition assay, the levels of hemagglutination (HA)-inhibiting (HI) antibodies in samples are determined as described previously (Palmer et al., 1975, Advanced laboratory techniques for influenza diagnosis. U.S. Department of Health, Education and Welfare Immunology Series. U.S. Department of Health, Education and Welfare, Washington, D.C.). Briefly, samples are incubated overnight at 37°C with 4 volumes of receptor-destroying 40 enzyme (Denka Seiken Co., Tokyo, Japan) prepared from Vibrio cholerae. After inactivation of the receptor-destroying enzyme by incubation of the samples at 56°C for 60 min, twofold serial dilutions of sera are mixed with 4 HA units of swine influenza virus. The assays are developed by adding 0.5% (vol/vol) chicken red blood cells, and the HI antibody titers are defined as the reciprocal of the highest dilution causing complete inhibition of agglutination.

45 5.9.5 Toxicity Studies

[0219] The toxicity and/or efficacy of the compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between 50 toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Therapies that exhibit large therapeutic indices are preferred. While therapies that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. [0220] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage 55 of the therapies for use in subjects (e.g., pigs). The dosage of such agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any therapy used in the method disclosed herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal

41 EP 2 497 492 B1

models to achieve a circulating plasma concentration range that includes the IC50 i.e.,( the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in subjects ( e.g., pigs). Levels in plasma may be measured, for example, by high performance liquid chromatography. 5 [0221] Further, any assays known to those skilled in the art can be used to evaluate the prophylactic and/or therapeutic utility of a composition, a combination therapy disclosed herein for swine influenza virus infection or a condition or symptoms associated therewith, an infection other than a swine influenza virus infection or a condition or symptom associated therewith, an IFN treatable disease, or a condition in which an attenuated swine influenza virus is used as a vector to induce an immune response to an antigen associated with the condition. 10 [0222] The following examples are provided to further illustrate the current invention but are not provided to in any way limit the scope of the current invention.

6. EXAMPLES

15 6.1 Example 1: Generation of Plasmid-derived Sw/Tx98 Viruses Encoding Truncated NS1

[0223] Site directed mutagenesis was used to generate different deletions in the swine influenza virus NS segment in such a way that the NEP was expressed without any alteration while the NS1 was partially deleted. This was achieved by inserting a stop codon in the NS1 ORF followed by a deletion in the NS1 ORF encompassing nucleotides not involved 20 in NEP expression. [0224] Cells and Viruses. Pig kidney-15 (PK-15), Swine Testis (ST) and Madin-Darby canine kidney (MDCK) type II cells were grown in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). Baby hamster kidney (BHK), 293T and A549 cells were grown in DMEM with 10% FBS. All cells were maintained at 37°C and 5%

CO2. The A/Swine/Texas/4199-2/98 (TX/98, H3N2 subtype) influenza virus was obtained from the repository at St. Jude 25 Children’s Research Hospital, Memphis, TN. Influenza viruses were grown in the allantoic cavities of embryonated chicken eggs or in MDCK cells. Recombinant vesicular stomatitis virus expressing GFP (VSV-GFP) (Stojdl, 2003, Cancer Cell 4:263-75) was grown and titrated in BHK cells for use in IFN bioassays. [0225] Construction of plasmids. MDCK cells were infected with TX/98 virus and total RNA was extracted by using Trizol reagent according to the manufacturer’s instructions (Invitrogen). The eight reverse genetics plasmids pHW-Sw- 30 PB2, pHW-Sw-PB1, pHW-Sw-PA, pHW-Sw-HA, pHW-Sw-NP, pHW-Sw-NA, pHW-Sw-M and pHW-Sw-NS (corresponding to the eight influenza viral segments listed in Table 1) were constructed by RT-PCR amplification of single viral RNA segments and cloning of the resulting cDNAs into the vector pHW2000 (Hoffmann et al, 2000, Proc. Natl. Acad. Sci. USA 97:6108-6113). The insert sequences for pHW-Sw-PB2, pHW-Sw-PB1, pHW-Sw-PA, pHW-Sw-HA, pHW-Sw-NP, pHW-Sw-NA, pHW-Sw-M and pHW-Sw-NS are provided in SEQ ID NOS: 1-8, respectively. In this system, influenza 35 viral cDNA is inserted between the RNA polymerase I (polI) promoter and terminator sequences. This entire RNA polI transcription unit is flanked by an RNA polymerase II (polII) promoter and a polyadenylation site. The orientation of the two promoters allows the synthesis of negative-sense viral RNAand positive-sense mRNAfrom one viral cDNA template. pHW-Sw-NS-73, pHW-Sw-NS-99 and pHW-Sw-NS-126 are derivatives of pHW-Sw-NS. These NS1 mutant plasmids were constructed by trimolecular ligation: Two PCR products were ligated into SalI/NgoIV digested pHW2000-Sw-NS. 40 [0226] The first PCR product was common for all three NS1 mutant plasmids, and was obtained using oligo pHW-3’ (reverse, annealing in the plasmid pHW2000 backbone) GGGTCAAGGAAGGCACGGGGGAGGGGC (SEQ ID NO: 9) and oligo 5’NS-PacI (forward, annealing in the NS 1 gene) GCGCTTAATTAAGAGGGAGCAATCGTTGGAG (SEQ ID NO: 10) and pHW2000-Sw-NS as template. This PCR product was digested with NgoIV and PacI. [0227] The second PCR product was specific for each NS 1 mutant plasmid, and was obtained using: a common 45 forward primer, CMV5’ (annealing in the CMV promoter of pHW2000 plasmid) GCCCCATTGACGCAAATGGGCGG- TAGGCGTG (SEQ ID NO: 11), and a specific reverse primer (annealing in the NS1 gene): NS73- BglII-PacI-3’ GCGCTTAATTAATCAAGATCTAGGATTCCTCTTTCAAAATCC (SEQ ID NO: 12), NS99-BglII-PacI-3’GCTTAATTAAT- CAAGATCTATGACATTTCCTCGAGGGTCATG (SEQ ID NO:13) or NS126-BglII-PacI-3 GCGCTTAATTAATCAA- GATCTACTTTTCCATGATCGCCTGGTCC (SEQ ID NO: 14). The three corresponding PCR products were digested 50 with SalI and PacI, and used in trimolecular ligation together with the PacI/NgoIV digested first PCR product and SaII/NgoIV digested pHW2000-Sw-NS, to generate: pHW-Sw -Tx-73, pHW-Sw-Tx-99 and pHW-Sw-Tx-126. These constructs contain a deletion in the NS1 sequence, plus the insertion of 4 stop codons in the 3 frames after this deletion. See Figure 1A. While the nuclear export protein (NEP) ORF is not altered in these constructs, the NS1 ORF encodes only the first 73, 99 and 126 amino acids of the wild-type NS1 protein (total length of wild type NS1 is 219 amino acids), 55 respectively. [0228] The viral strains are described below:

Sw/TX/98 wt: contains wild-type NS1 gene and represents a triple-reassortant H3N2 swine influenza virus.

42 EP 2 497 492 B1

Sw/TX/98/del126: expresses the N-terminal 126 amino acids of the wild-type NS1 protein. Sw/TX/98/del99: expresses the N-terminal 99 amino acids of the wild-type NS1 protein. Sw/TX/98/del73: expresses the N-terminal 72 amino acids of the wild-type NS1 protein.

5 [0229] Transfection-mediated recovery of recombinant SIV. Rescue of influenza viruses from plasmid DNA was performed as described in Fodor et al. (1999, J Virol 73:9679-82) and Neumann et al. (1999, Proc NatI Acad Sci USA 96:9345-50) using an eight-plasmid system (Hoffmann et al., 2000, Proc. Natl. Acad. Sci. USA 97:6108-6113). To generate the recombinant wild type (rWT) TX/98 virus 0.5 mg of each of the 8 pHW plasmids were transfected into 10 6 293T cells in suspension using the Lipofectamine 2000 reagent (Invitrogen). the NS1 truncated mutant viruses were 10 generated in the same way but substituting the pHW-Sw-NS plasmid by the corresponding mutant one to recover the del73, del99 or del126 virus mutants. The resulting viruses were passaged and cloned by plaque purification into MDCK cells. Virus stocks were grown in 7-day-old embryonated chicken eggs. The identity of the recombinant viruses SIV was verified by gel electrophoretic analyses of RT-PCR products derived from viral RNA using oligonucleotides specific for the common non-coding regions. The wild type NS gene or the deleted versions were further confirmed by sequencing. 15 [0230] Results. To generate SIV using reverse genetics, the eight viral RNAs from Tx/98 virus were cloned into ambisense expression plasmids for viral rescue (Hoffmann et al., 2000, Proc. Natl. Acad. Sci. USA 97:6108-6113). Upon transfection of these plasmids in 293T cells, infectious viruses were recovered. The parental (WT) and the plasmid- derived wild type (rWT) SIVs grew to similar titers in MDCK cells and embryonated eggs. When inoculated into pigs, the rWT virus produced similar lesions as the parental WT virus. Three NS1-truncated SIV mutants were generated encoding 20 NS1 proteins of 73, 99 and 126 amino acids, as compared to the full length, 219 amino acids long, NS1 protein (Fig. 1A). RT-PCR and sequencing analyses confirmed the presence of the truncated NS1 genes in the rescued virus preparations (Fig. 1B).

6.2 Example 2: Characterization of the Swine Influenza Virus NS1 Deletion Mutants and Wild Type A/Swine/Tex- 25 as/4199-2/98

[0231] To characterize the impact of the NS1 gene deletion on the replicative properties of swine influenza virus, the growth of the wild-type and the different deletion mutants described in Section 6.1 was compared. Also, the effect of these mutants on IFN production was compared. 30 [0232] Virus growth curves. To analyze viral replication, confluent PK-15 cells were infected at the indicated multiplicity of infection (MOI) and incubated for different periods of times at 37°C in MEM containing 0.3% bovine albumin (MEM/BA) and 5% allantoic fluid. Virus titers were determined by plaque assay on MDCK cells in MEM/BA supplemented with 1 mg/ml of TPCK trypsin. Titers were expressed as plaque forming units (PFU) per ml. [0233] In order to investigate the multicycle growth properties of the mutant NS 1 viruses confluent pig epithelial cells 35 (PK-15) were infected at a low MOI (MOI=0.001). Supernatants from infected cells were titrated at different time points post infection by plaque assay on MDCK cells. The growth kinetics of the NS 1 deletion mutants in PK-15 cells were clearly different when compared to the wild type virus. Interestingly, the 1-126 mutant virus was the most compromised in growth, followed by the 1-99 and the 1-73 mutants (Fig. 2A). Plaque sizes in MDCK cells correlated with the growth differences in PK-15 cells (Fig 2C). These results indicate that deletions in the NS1 protein of Sw/TX/98 virus result in 40 attenuation of viral growth, both in PK-15 and MDCK cells. In contrast, when NS1 mutant viruses were grown at a high MOI (MOI=2) in PK-15 cells major differences in growth kinetics were not detected (Fig. 2B). This most likely indicates that virus replication of the NS1 mutant viruses is not restricted during the first cycle of replication, suggesting that infected cells secrete antiviral cytokines, such as IFN, that inhibit subsequent rounds of replication. [0234] Metabolic labeling. Confluent PK-15 cells seeded in 22-mm dishes were either mock-infected or infected with 45 rWT, 1-73, 1-99 or 1-126 viruses at an MOI of 2. Cells were incubated in MEM/BA at 37°C for various time points, and subsequently labeled for 2 h with 10m Ci [35S]Met-Cys in MEM lacking Met-Cys. Cells were washed with ice-cold phosphate-buffered saline (PBS), lysed and total cell extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were visualized by autoradiography. [0235] Metabolic labeling of virus-infected PK cells (MOI=2) with [ 35S]-Met-Cys, revealed no differences in the kinetics 50 of NP protein synthesis among rWT and 1-73, 1-99 and 1-126 mutant viruses (Fig. 2D). The NS1 mutant proteins could not be detected by labeling. [0236] Bioassay to measure IFN production. Levels of IFN secreted by virus infected cells were determined as previously described (Park et al., 2003, J. Virol. 70:9522-9532; Donelan, 2003, J Virol 77:13257-66), with some variations. Confluent PK-15 cells seeded in 22-mm dishes were either mock-treated or infected with rWT, del73, del99 or del126 55 viruses at an MOI of 2. Following infection, cells were incubated with MEM/BA containing 5% allantoic fluid, and at differenttime points post-infection, supernatants were harvested. Viruses present in the supernatant were UV inactivated by placing samples on ice 6 inches below an 8-W UV lamp (Fisher) for 15 min with constant stirring. New PK-15 cells were seeded in 96-well plates the day before and incubated with the UV-inactivated supernatants for 24 h. The prein-

43 EP 2 497 492 B1

cubated PK-15 cells were then infected with VSV-GFP (MOI=0.1). The cells expressing GFP were visualized by fluorescence microcopy 16 hours post infection. [0237] In order to see whether the observed attenuation of growth in vitro of the NS1-truncated viruses was directly correlated with the ability of these viruses to inhibit the IFN-α/β system, the induction of IFN in cells infected with the 5 recombinant Sw/Tx/98 viruses was investigated. Supernatants of infected PK-15 cells were used to determine the levels of secreted IFN-α/β using a bioassay based on inhibition of VSV-GFP replication (Fig. 3A). For this purpose, PK-15 cells were infected (MOI=2) with rWT and NS1 mutant SIVs and supernatants were collected for IFN determinations every two hours for 10 h. The results are shown in Fig. 3B. Supernatants from mock-infected cells caused no inhibition of GFP expression by VSV-GFP in PK-15 cells. In contrast, VSV-GFP replication was completely abolished in cells pretreated 10 with the supernatant of del126 virus-infected cells at 10 h post infection. No IFN-α/β was detected by this assay in supernatants of cells infected with rWT virus. All NS1-truncated viruses induced detectable levels of IFN- α/β by 6 h post infection, with 1-126 virus being the stronger IFN- α/β inducer, followed by del99 and del73 viruses. Similar experiments were performed using other virus-infected swine (ST) and human (A549) cell lines with basically identical results. [0238] Analysis of IFN-β and TNF-α mRNA by RT-PCR. PK-15 cells were infected at an MOI of 2, and at 24 hours 15 post infection, total RNA was extracted using Absolutely RNA RT-PCR Miniprep Kit (Stratagene). RT was performed by using oligodT as the primer. PCR was done using specific pairs of primers for swine IFN-β (5-SWIFNB+ GGCCAT- GGCTAACAAGTGCATCC (SEQ ID NO: 15), 3-SWIFNB-CCGGTCAGTTCCGGAGGTAATC (SEQ ID NO: 16)) and TNF-α (5’SW-TNFAATGAGCACTGAGAGCATG (SEQ ID NO: 17), 3’SW-TNFATCACAGGGCAATGATCCC (SEQ ID NO: 18)) mRNAs (Genbank accession numbers M86762 and X57321). As control, specific primers for p-actin (Donelan, 20 2003 J Virol 77:13257-66) were used to amplify a 550-bp fragment of swine p-actin. The products were sequenced and confirmed to be derived from the expected mRNAs. [0239] RT-PCR analysis of IFN-β- and TNF-α- transcripts revealed the induction of the expression of the mRNA of these cytokines in PK-15 cells infected with NS1 mutant viruses, especially in 1-126 virus-infected cells (Fig. 3C). These data indicate that the potency of inhibition of IFN production by the recombinant Sw/Tx/98 viruses is as follows: 25 rWT>del73>del99>del126.

6.3 Example 3: Pathogenesis Of Plasmid-Derived TX/98 Mutant/Deletion Viruses

[0240] Pathogenesis of the specific swine influenza viruses described in Section 6.1 were assessed in pigs. The ability 30 of Sw/Tx/98/del126, Sw/Tx/98/del99, and Sw/Tx/98/del73 to induce lung lesions and cause infections in swine were determined and compared to the results obtained for a mock virus and the wild-type virus Sw/Tx/98 wt. Lung lesions were assessed as the percentage of the healthy lung in which lesions occurred with an average of seven lobes of the lung being used for this determination. Viruses or the mock were administered as described in detail below and either nasal swabs or BALF were obtained during necropsy to determine virus burden five days post infection. Histopathologic 35 evaluation was preformed. The nasal turbinates and trachea were examined for epithelial changes and subepithelial inflammation. The alveoli were also evaluated for inflammatory changes. [0241] This example demonstrates that C-terminal deletions of the NS1 gene result in an attenuated phenotype.

6.3.1 Methods 40 [0242] Infection of pigs. Out-bred specific pathogen-free pigs were obtained from a commercial hog farm in Iowa (USA): Sera from these pigs was confirmed to be negative for the presence of hemagglutination inhibition antibodies (Palmer, 1975, (U.S. Department of Health, Education, and Welfare, Washington, D.C.), pp. 51-52) against H3N2 (Sw/TX/98 and Sw/CO/99) and H1N1 (Sw/IA/30) SIV. Groups of four to five pigs were housed in separate isolation 45 rooms. At the age of 4 weeks, pigs were anesthetized by intramuscular injection with a mixture of ketamine, xylazine, zolazepam and tiletamine (Mengeling, 1996, Am J Vet Res 57:834-839) before being inoculated intratracheally (via laryngoscope and tracheal tube) with 1 ml of virus solution containing 1x105 PFU. For 5 days, pigs were monitored for clinical signs including lethargy, anorexia, coughing, hyperpnea/dyspnea, nasal and ocular discharge and their body temperature was recorded daily. At day 5, animals were euthanized by intravenous administration of pentobarbital (Fort 50 Dodge Animal Health, Fort Dodge’, USA), and their lungs were removed in toto. The percentage of the surface of each pulmonary lobe that was affected by macroscopic lesions was estimated visually. The percentages were averaged to obtain a mean value for the 7 pulmonary lobes of each animal. The nasal turbinates, trachea, and right cardiac lobe were removed and formalin-fixed for further histopathologic evaluation. Blood, broncho-alveolar fluid (BALF) and nasal swabs were also collected during necropsy. BALF was obtained after the lungs were removed from the thoracic cavity 55 by rinsing the lungs (via trachea) with ca. 30 ml of McCoy’s medium without serum. All animal experiments were in compliance with the Institutional Animal Care and Use Committee of the National Animal Disease Center. [0243] Clinical observation and sampling. For 5 days, pigs were monitored for clinical signs including lethargy, anorexia, coughing, hyperpnea/dyspnea, nasal and ocular discharge and their body temperature was recorded daily.

44 EP 2 497 492 B1

Blood, BALF, and nasal swabs were collected during necropsy. BALF was obtained after the lungs were removed from the thoracic cavity by rinsing the lungs (via trachea) with ca. 30 ml of McCoy’s medium without serum. Nasal swabs and BALF were tested to determine virus burden. [0244] Virus titrations from nasal swabs and BALF. Nasal swabs and BALF were tested to determine virus burden. 5 Ten-fold serial dilutions were prepared in McCoy’s medium without serum, supplemented with 5 mg/ml trypsin. MDCK cells were inoculated with the dilutions and incubated with medium plus trypsin in microliter plates at 37 °C for 72 h. The plates were examined for cytopathic effects after 72 hours. Virus titers were calculated by the Reed and Muench method (Reed, 1938, Am J Hyg 27:493-497). [0245] Histopathological evaluation: The nasal turbinates and trachea were strained with hematoxylin and eosin 10 and examined under the microscope for epithelial changes and subepithelial inflammation. The lungs were examined for bronchiolar epithelial changes and peribronchiolar inflammation in large, medium, and small or terminal bronchioles. The alveoli were also evaluated for inflammatory changes. Because lesions were found most consistently in medium- sized airways, data obtained from the medium bronchioles were used for comparisons. The medium bronchioles were graded on a scale of 0 to 3+ as follows: 0 (normal: lined by medium to tall columnar epithelial cells with ciliated apical 15 borders and basal pseudostratified nuclei; minimal inflammation); 1+ (epithelial layer columnar and even in outline with only slightly increased proliferation; cilia still visible on many cells); 2+ (prominent changes in the epithelial layer rangin g from attenuation to marked proliferation; cells disorganized and layer outline irregular at the luminal border); 3+ (epithelial layer markedly disrupted and disorganized with necrotic cells visible in the lumen; some bronchioles attenuated and others in marked reactive proliferation). 20 [0246] The trachea was graded on a scale of 0 to 2.5+ as follows: 0 (normal: Lined by medium to tall columnar epithelial cells with ciliated apical border, nuclei basal and pseudostratified. Cytoplasm evident between apical border and nucleus. Occasional small focus with squamous cells); 1+: (focal squamous metaplasia of the epithelial layer); 2+ (diffuse squamous metaplasia of much of the epithelial layer, cilia may be evident focally); 2.5+ (diffuse squamous metaplasia with very few cilia evident). 25 [0247] SIV immunohistochemistry was performed using mouse NP-specific monoclonal antibody HB65 (American Type Culture Collection, Manassas, Virginia). The antibody was produced as mouse ascites fluid. A dilution of 1/1000. of HB65 was used for immunohistochemistry. The assay was performed using the Dako Envision IHC system.. Staining was graded 0 to 3+ as follows: 0 (no infected cells); 0.5+ (few infected cells); 1+ (few infected cells, as widely separated individual cells); 1.5+ (few infected cells, as widely separated singles and in small clusters); 2+ (moderate numbers of 30 infected cells, usually affecting clusters of adjacent cells in portions of the epithelial layer lining bronchioles, or in smal l sublobular foci in alveoli); 3+ (numerous infected cells, affecting most of the epithelial layer in bronchioles, or widespread in large sublobular foci in alveoli). [0248] Statistical analysis. Mean body temperatures, extent of gross and histopathologic changes, and virus replication in infected and control groups were compared by using the two-sided Student’s t-test. Probab ility (P) values <0.05 35 were considered to indicate a statistically significant difference between groups.

6.3.2 Results

[0249] Forty seven 4-week-old outbred pigs were purchased from a commercial hog farm in lowa. Groups of 10 pigs 40 were intratracheally infected with 10 5 pfu of rWT and NS 1 mutant Sw/TX/98 viruses. Seven animals were mock-infected with medium only. The mean rectal temperature of each infected group increased to ≥40 °C 1 to 3 days p.i. in the rWT, 1-73 and 1-99 virus-infected cohorts (data not shown). Not all of the animals infected with NS1 deletion mutant 1-126 exhibited increased temperature. Clinical signs, which comprised respiratory distress, nasal secretion, conjunctivitis, and coughing began on days 2 to 4 p.i. in the rWT-virus infected group. The prevalence of clinical signs differed between 45 the respective viruses. Most clinical signs were consistently observed in the groups infected with the rWT virus while some signs were observed in the 1-99 and 1-73 virus-infected cohorts. Only elevated temperature but no other clinical signs were observed in the 1-126 virus-infected pigs. [0250] During necropsy at day 5 p.i., the percentage of each lung surface with macroscopic lesions was estimated. As shown in Fig. 4A, mock-infected control animals had no macroscopic lung lesions. Pigs infected with the rWT Sw/Tx/98 50 virus showed significantly higher percentage of macroscopic lung lesions than pigs infected with the NS1 deletion mutant viruses 1-99 (p=0.006) or 1-126 (p=0.004). Pigs infected with the NS 1 deletion mutant virus 1-73 displayed less severe lesions, however, this was not statistically not significant (p>0.05). In general, the gross lesions we observed were marked, plum-colored, consolidated areas on individual lobes. The diaphragmatic lobes were less involved than the other lobes. The mediastinal lymph nodes were usually hyperemic and enlarged. 55 [0251] The medium bronchioles were microscopically examined in tissue sections of the right cardiac pulmonary lobe of 4-week-old infected pigs and were histopathologically scored (Fig. 4B). Mock-infected animals as well as 1-126 virus- infected pigs, showed no or minimal lesions, whereas moderate to severe lesions were detected in animals infected with the rWT and the 1-99 and 1-73 Sw/TX/98 viruses (Figs. 5A-D and 6A-D). All animals infected with the parental wild-

45 EP 2 497 492 B1

type and some infected with the 1-73 and 1-99 viruses had high scores reflecting disruption of the bronchial epithelial cell layer (characterized by acute epithelial necrosis or subsequent attenuation or reactive proliferation). Most animals infected with either 1-73 or 1-99 virus had a moderate score reflecting slightly increased proliferation of the bronchial epithelial layer. The lungs of animals infected with the NS1 deletion mutant 1-126 virus were nearly devoid of lesions. 5 As in the macroscopic lung lesions, 1-126 and 1-99 infected pigs showed significantly less damage than rWT-infected pigs (1-126: p<0.0001; 1-99: p=0.0002). Interestingly, microscopic lesions in bronchioles of pigs infected with the 1-73 mutant virus were also significantly less when compared to the parental rWT virus (p=0.02). [0252] Virus titers in the BALF were also analyzed at day 5 p.i. As shown in Fig. 4C, all viruses replicated in the respiratory tract of pigs. BALF virus titers were significantly higher in animals infected with parental rWT virus when 10 compared to the NS1 deletion mutants. This finding correlates with significantly less extensive gross and microscopic disease observed in pigs infected with NS1 deletion mutants versus wild-type Sw/Tx/98 viruses.

6.4 Example 4: Use of TX/98/del 126 as a vaccine

15 [0253] The efficacy of the attenuated TX/98/del 126 deletion mutant was tested in a pig vaccination study. Experiments were performed as described in Example 3, except that vaccination with TX/98/del 126 was performed twice on days 0 and day 21 with 1x10 5 PFU per pig by intratracheal inoculation. The vaccination and challenge was done by intratracheal inoculation (using 1 ml of virus solution/ or medium as a control). Animals were challenged 9 days or 10 days later with the following preparations: 20 • 2x105 PFU per pig with wild-type H3N2 virus (A/Swine/Texas/4199-2/98-homologous challenge) • 2x105 PFU per pig with wild-type H1N1 virus (A/Swine/MN/37866/99 classical H1N1 - heterologous challenge) • Mock challenge with 1 ml medium

25 [0254] For histopathological evaluation, the right cardiac lobe of each lung was stained with hematoxylin and eosin and examined for bronchiolar epithelial changes and peribronchiolar inflammation in large, medium, and small or terminal bronchioles. Because lesions were found most consistently in medium-sized airways, data obtained from the medium bronchioles were used for comparisons. Lesion severity was scored by the distribution or extent of lesions within the sections examined as follows: 0 - No airways affected. 1 - Only a few isolated airways affected; 2- Localized cluster of 30 affected airways, often within one or two lobules. 3 - Low number of airways affected but throughout much of the section. 4 - Many airways affected, often severely, all sizes. [0255] One trained examiner was utilized for evaluation of tissue sections. The examiner was unaware of which group of animals the tissues were derived from. [0256] Immunohistochemistry was done using a monoclonal antibody specific for the nucleoprotein of influenza. The 35 antigen score was as follows:

0= No antigen positive cells. 1=Only a few cells with positive staining in an occasional airway. 2=Only a few cells with positive staining in scattered airways which may be localized. 40 3=Moderate numbers of cells with positive staining in an occasional airway. 4=Moderate numbers of cells with positive staining in scattered airways and alveoli.

[0257] Lung lavage was tested to determine virus load. Ten-fold serial dilutions were prepared in McCoy’s medium without serum, supplemented with 5 mg/ml trypsin. MDCK cells were inoculated with the dilutions and incubated with 45 medium plus trypsin in microtiter plates at 37 °C for 72 h. The plates were examined for cytopathic effects after 72 hours. Virus titers were calculated by the Reed and Muench method. [0258] Results. The following results were obtained 5 days after challenge:

1. Nonvaccinated, nonchallenged pigs did not have any histopathological changes and did not harbor influenza 50 virus antigen in their lung tissues (Figs. 7 and 8). Infectious virus was not present or below detection limits (Fig. 9). 2. Nonvaccinated pigs challenged with wild type H3N2 TX/98 virus showed significant histopathological damage 6.28 and influenza viral antigen in their lung tissues (Figs. 7 and 8). Virus titers in lung lavage was ≥10 TCID50/ml. 3. Nonvaccinated pigs challenged with wild type H1N1 MN/99 virus showed significant histopathological damage 6.0 and influenza viral antigen in their lung tissues (Figs. 7 and 8). Virus titers in lung lavage was ≥10 TCID50/ml. 55 4. Vaccinated, mock challenged pigs did not have any histopathological changes and did not harbor influenza virus antigen in their lung tissues (Figs. 7 and 8). Infectious virus was not present or below detection limits (Fig. 9). 5. Vaccinated, H3N2 challenged pigs did not have any histopathological changes and did not harbor influenza virus antigen in their lung tissues (Figs. 7 and 8). Infectious virus was not present or below detection limits (Fig 9).

46 EP 2 497 492 B1

6. Vaccinated, H1N1 challenged pigs did have significantly less histopathological changes in their lung tissues compared to nonvaccinated, H1N1 challenged pigs (p<0.001). These pigs did not harbor influenza virus antigen in their lung tissues (Figs. 7 and 8). There was significantly less (p<0.001) infectious virus present in the lung lavage when compared to the nonvaccinated H1N1 challenged animals (Fig. 9). 5 [0259] In summary, vaccination of pigs with the attenuated TX/98/del 126 mutant resulted in protective immunity against challenge with a homologous virus isolate (H3N2 A/Swine/Texas/4199-2/98 virus challenge). When vaccinated pigs were challenged with a virus belonging to a different influenza subtype (H1N1 A/Swine/MN/37866/99 virus challenge), this heterologous virus challenge resulted in significant less lesions in lung tissues and virus load in lung lavage 10 when compared to the nonvaccinated controls at day 5 post inoculation. [0260] These data indicate that attenuated TX/98/del 126 mutant has utility as a modified live virus vaccine showing protective immunity against homologous/ homotypic challenge and considerable protection against heterologous/ het- erotypic challenge.

15 SEQUENCE LISTING

[0261]

<110> Mount Sinai school of Medicine of New York University 20 St. Jude children’s Research Hospital The United States of America as represented by the secretary of Agriculture

<120> Genetically Engineered Swine influenza viruses and Uses Thereof

25 <130> 6923-116-228

<150> 60/576,418 <151> 2004-06-01

30 <160> 18

<170> FastSEQ for Windows version 4.0

<210> 1 35 <211> 2318 <212> DNA <213> Artificial Sequence

<220> 40 <223> plasmid insert PB2

<400> 1

47 EP 2 497 492 B1

35 <210> 2 <211> 2348 <212> DNA <213> Artificial Sequence 40 <220> <223> plasmid insert PB1

<400> 2 45

48 EP 2 497 492 B1

35 <210> 3 <211> 2240 <212> DNA <213> Artificial sequence 40 <220> <223> plasmid insert PA

<400> 3 45

49 EP 2 497 492 B1

<210> 4 <211> 1769 <212> DNA 40 <213> Artificial sequence

<220> <223> plasmid insert HA

45 <400> 4

50 EP 2 497 492 B1

<210> 5 <211> 1572 30 <212> DNA <213> Artificial Sequence

<220> <223> plasmid insert NP 35 <400> 5

51 EP 2 497 492 B1

15 <210> 6 <211> 1453 <212> DNA <213> Artificial sequence

20 <220> <223> plasmid insert NA

<400> 6

<210> 7 <211> 1032 50 <212> DNA <213> Artificial sequence

<220> <223> plasmid insert M 55 <400> 7

52 EP 2 497 492 B1

20 <210> 8 <211> 873 <212> DNA <213> Artificial sequence

25 <220> <223> NS

<400> 8

<210> 9 45 <211> 27 <212> DNA <213> Artificial sequence

<220> 50 <223> oligo pHW-3’ (reverse) for annealing in the plasmid pHW2000 backbone

<400> 9 gggtcaagga aggcacgggg gaggggc 27

55 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence

53 EP 2 497 492 B1

<220> <223> oligo 5’NS-PacI for annealing in the NS1 gene

<400> 10 5 gcgcttaatt aagagggagc aatcgttgga g 31

<210> 11 <211> 31 <212> DNA 10 <213> Artificial sequence

<220> <223> primer CMV5’ annealing in the CMV promoter of pHW2000 plasmid

15 <400> 11 gccccattga cgcaaatggg cggtaggcgt g 31

<210> 12 <211> 42 20 <212> DNA <213> Artificial sequence

<220> <223> reverse primer NS73- BglII-PacI-3’ annealing in the NS1 gene 25 <400> 12 gcgcttaatt aatcaagatc taggattcct ctttcaaaat cc 42

<210> 13 30 <211> 42 <212> DNA <213> Artificial Sequence

<220> 35 <223> primer NS99-BglII-PacI-3’

<400> 13 gcttaattaa tcaagatcta tgacatttcc tcgagggtca tg 42

40 <210> 14 <211> 44 <212> DNA <213> Artificial sequence

45 <220> <223> primer NS126-BglII-PacI-3’

<400> 14 gcgcttaatt aatcaagatc tacttttcca tgatcgcctg gtcc 44 50 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence 55 <220> <223> specific primer 5-SWIFNB+ for swine IFN-beta

54 EP 2 497 492 B1

<400> 15 ggccatggct aacaagtgca tcc 23

<210> 16 5 <211> 22 <212> DNA <213> Artificial Sequence

<220> 10 <223> specific primer 3-SWIFNB- for swine IFN-beta

<400> 16 ccggtcagtt ccggaggtaa tc 22

15 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence

20 <220> <223> primer 5’SW-TNFA for NF-a (see Genbank accession No. M86762)

<400> 17 atgagcactg agagcatg 18 25 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence 30 <220> <223> primer 3’SW-TNFA (see Genbank accession No. X57321)

<400> 18 35 tcacagggca atgatccc 18

Claims

40 1. A genetically engineered attenuated swine influenza virus, wherein the virus comprises a swine influenza virus NS1 gene with a mutation resulting in a swine influenza virus NS1 protein having a deletion of exactly 90 amino acid residues from the carboxy-terminus of NS1, wherein the swine influenza virus NS1 gene is from A/Swine/Tex- as/4199-2/98.

45 2. The genetically engineered attenuated swine influenza virus of claim 1, wherein the swine influenza virus is H1N1, H1N2, or H3N2.

3. The genetically engineered attenuated swine influenza virus of claim 1, wherein the attenuated virus is a reassortant.

50 4. The genetically engineered attenuated swine influenza virus of claim 1, wherein the swine influenza virus is A/Swine/Colorado/1/77, A/Swine/Colorado/23619/99, A/Swine/Cote d’Armor/3633/84, A/Swine/Eng- land/195852/92, A/Swine/Finistere/2899/82, A/Swine/Hong Kong/10/98, A/Swine/Hong Kong/9/98, A/Swine/Hong Kong/81/78, A/Swine/Illinois/100084/01, A/Swine/Illinois/100085A/01, A/Swine/Illinois/21587/99, A/Swine/Indi- ana/1726/88, A/Swine/Indiana/9K035/99, A/Swine/Indiana/P12439/00, A/Swine/Iowa/30, A/Swine/Iowa/15/30, 55 A/Swine/Iowa/533/99, A/Swine/Iowa/569/99, A/Swine/Iowa/3421/90, A/Swine/Iowa/8548-1/98, A/Swine/Io- wa/930/01, A/Swine/Iowa/17672/88, A/Swine/Italy/1513-1/98, A/Swine/Italy/1523/98, A/Swine/Korea/CY02/02, A/Swine/Minnesota/55551/00, A/Swine/Minnesota/593/99, A/Swine/Minnesota/9088-2/98, A/Swine/Nebras- ka/1/92, A/Swine/Nebraska/209/98, A/Swine/Netherlands/12/85, A/Swine/North Carolina/16497/99, A/Swine/North

55 EP 2 497 492 B1

Carolina/35922/98, A/Swine/North Carolina/93523/01, A/Swine/North Carolina/98225/01, A/Swine/Oeden- rode/7C/96, A/Swine/Ohio/891/01, A/Swine/Oklahoma/18717/99, A/Swine/Oklahoma/18089/99, A/Swine/On- tario/01911-1/99, A/Swine/Ontario/01911-2/99, A/Swine/Ontario/41848/97, A/Swine/Ontario/97, A/Swine/Que- bec/192/81, A/Swine/Quebec/192/91, A/Swine/Quebec/5393/91, A/Swine/Taiwan/7310/70, A/Swine/Tennes- 5 see/24/77, A/Swine/Texas/4199-2/98, A/Swine/Wisconsin/125/97, A/Swine/Wisconsin/136/97, A/Swine/Wiscon- sin/163/97, A/SwinelWisconsin/164/97, A/Swine/Wisconsin/166/97, A/Swine/Wisconsin/168/97, A/Swine/Wiscon- sin/235/97, A/Swine/Wisconsin/238/97, A/Swine/Wisconsin/457/98, A/Swine/Wisconsin/458/98, A/Swine/Wiscon- sin/464/98 or A/Swine/Wisconsin/14094/99.

10 5. The genetically engineered attenuated swine influenza virus of claim 4, wherein the swine influenza virus is A/Swine/Texas/4199-2/98.

6. The genetically engineered attenuated swine influenza virus of any one of claims 1 to 5, wherein the attenuated virus is a chimeric virus that expresses a heterologous sequence. 15 7. The genetically engineered attenuated swine influenza virus of any one of claims 1 to 5, wherein the attenuated virus is a chimeric virus that expresses an epitope of a foreign pathogen.

8. The genetically engineered attenuated swine influenza virus of any one of claims 1 to 5, wherein the attenuated 20 virus is a chimeric virus that expresses a tumor antigen.

9. The genetically engineered attenuated swine influenza virus of any one of claims 1 to 5, wherein the attenuated virus is engineered to express an epitope from a different virus or at least one segment derived from a different virus.

25 10. An immunogenic formulation comprising the genetically engineered attenuated swine influenza virus of any one of claims 1 to 9, and a physiologically acceptable excipient.

11. The immunogenic formulation of claim 10, wherein the concentration of the attenuated virus in said immunogenic formulation is about 104 to about 107 pfu/ml. 30 12. A pharmaceutical composition comprising the genetically engineered attenuated swine influenza virus of any one of claims 1 to 9, and a physiologically acceptable excipient.

13. The pharmaceutical composition of claim 12, wherein the concentration of the attenuated virus in said pharmaceutical 35 composition is about 104 to about 1012 pfu/ml.

14. A genetically engineered attenuated swine influenza virus as defined in any one of claims 1 to 9 for use in immunizing or inducing an immune response in a pig.

40 15. A genetically engineered attenuated swine influenza virus as defined in any one of claims 1 to 9 for use in the prevention of a disease or symptom associated with swine influenza virus infection in a pig.

16. A genetically engineered attenuated swine influenza virus as defined in any one of claims 1 to 9 for use in the treatment of a disease associated with a swine influenza virus infection in a pig. 45 17. An immunogenic formulation as defined in claim 10 or 11 for use in immunizing or inducing an immune response in a pig.

18. A pharmaceutical composition as defined in claim 12 or 13 for use in the prevention of a disease or symptom 50 associated with swine influenza virus infection in a pig.

19. A pharmaceutical composition as defined in claim 12 or 13 for use in the treatment of a disease associated with a swine influenza virus infection in a pig.

55 20. A cultured cell containing the genetically engineered attenuated swine influenza virus of any one of claims 1 to 9.

21. The cultured cell of claim 20, wherein the cell is a pig cell or pig cell line.

56 EP 2 497 492 B1

22. The cultured cell of claim 21, wherein the pig cell line is PK(D1) cells, PK(15) cells, PK13 cells, SJPL cells, NSK cells, LLC-PK1 cells, LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells or PK-2a/CL 13 cells.

23. An embryonated egg containing the genetically engineered attenuated swine influenza virus of any one of claims 5 1 to 9.

24. The embryonated egg of claim 23 which is from a chicken.

25. The embryonated egg of claim 23 or 24, wherein the embryonated egg is 6, 7, or 8 days old. 10 26. The cultured cell of any one of claims 20 to 22, or the embryonated egg of any one of claims 23 to 25, wherein the cultured cell or the embryonated egg is interferon deficient.

27. A method for production of an immunogenic formulation or a pharmaceutical formulation comprising: 15 (a) propagating the genetically engineered attenuated swine influenza virus of any one of claims 1 to 9 in

(i) the cell of any one of claims 20 to 22 or 26, or (ii) the embryonated egg of any one of claims 23 to 26, and 20 (b) collecting the progeny virus.

Patentansprüche 25 1. Genetisch verändertes abgeschwächtes Schweinegrippe-Virus, wobei das Virus ein Schweinegrippe-Virus NS1- Gen mit einer Mutation umfasst, die zu einem Schweinegrippe-Virus NS1 Protein führt, das eine Deletion von genau 90 Aminosäureresten ab dem NS1 Carboxy-Terminus hat, wobei das Schweinegrippe-Virus NS1-Gen von A/Swine/Texas/4199-2/98 ist. 30 2. Genetisch verändertes abgeschwächtes Schweinegrippe-Virus nach Anspruch 1, wobei das Schweinegrippe-Virus H1N1, H1N2 oder H3N2 ist.

3. Genetisch verändertes abgeschwächtes Schweinegrippe-Virus nach Anspruch 1, wobei das abgeschwächte Virus 35 ein Reassortant ist.

4. Genetisch verändertes abgeschwächtes Schweinegrippe-Virus nach Anspruch 1, wobei das Schweinegrippe-Virus A/Swine/Colorado/1/77, A/Swine/Colorado/23619/99, A/Swine/Cote d’Armor/3633/84, A/Swine/Eng- land/195852/92, A/Swine/Finistere/2899/82, A/Swine/Hong Kong/10/98, A/Swine/Hong Kong/9/98, A/Swine/Hong 40 Kong/81/78, A/Swine/Illinois/100084/01, A/Swine/Illinois/100085A/01, A/Swine/Illinois/21587/99, A/Swine/India- na/1726/88, A/Swine/Indiana/9K035/99, A/Swine/Indiana/P12439/00, A/Swine/Iowa/30, A/Swine/Iowa/15/30, A/Swine/Iowa/533/99, A/Swine/Iowa/569/99, A/Swine/Iowa/3421/90, A/Swine/lowa/8548-1/98, A/Swine/Io- wa/930/01, A/Swine/Iowa/17672/88, A/Swine/Italien/1513-1/98, A/Swine/Italien/1523/98, A/Swine/Korea/CY02/02, A/Swine/Minnesota/55551/00, A/Swine/Minnesota/593/99, A/Swine/Minnesota/9088-2/98, A/Swine/Nebras- 45 ka/1/92, A/Swine/Nebraska/209/98, A/Swine/Niederlande/12/85, A/Swine/North Carolina/16497/99, A/Swine/North Carolina/35922/98, A/Swine/North Carolina/93523/01, A/Swine/North Carolina/98225/01, A/Swine/Oedenro- de/7C/96, A/Swine/Ohio/891/01, A/Swine/Oklahoma/18717/99, A/Swine/Oklahoma/18089/99, A/Swine/Onta- rio/01911-1/99, A/Swine/Ontario/01911-2/99, A/Swine/Ontario/41848/97, A/Swine/Ontario/97, A/Swine/Que- bec/192/81, A/Swine/Quebec/192/91, A/Swine/Quebec/5393/91, A/Swine/Taiwan/7310/70, A/Swine/Tennes- 50 see/24/77, A/Swine/Texas/4199-2/98, A/Swine/Wisconsin/125/97, A/Swine/Wisconsin/136/97, A/Swine/Wiscon- sin/163/97, A/Swine/Wisconsin/164/97, A/Swine/Wisconsin/166/97, A/Swine/Wisconsin/168/97, A/Swine/Wiscon- sin/235/97, A/Swine/Wisconsin/238/97, A/Swine/Wisconsin/457/98, A/Swine/Wisconsin/458/98, A/Swine/Wiscon- sin/464/98 oder A/Swine/Wisconsin/14094/99 ist.

55 5. Genetisch verändertes abgeschwächtes Schweinegrippe-Virus nach Anspruch 4, wobei das Schweinegrippe-Virus A/Swine/Texas/4199-2/98 ist.

6. Genetisch verändertes abgeschwächtes Schweinegrippe-Virus nach einem der Ansprüche 1 bis 5, wobei das ab-

57 EP 2 497 492 B1

geschwächte Virus ein chimäres Virus ist, das eine heterologe Sequenz exprimiert.

7. Genetisch verändertes abgeschwächtes Schweinegrippe-Virus nach einem der Ansprüche 1 bis 5, wobei das ab- geschwächte Virus ein chimäres Virus ist, das ein Epitop eines fremden Pathogens exprimiert. 5 8. Genetisch verändertes abgeschwächtes Schweinegrippe-Virus nach einem der Ansprüche 1 bis 5, wobei das ab- geschwächte Virus ein chimäres Virus ist, das ein Tumorantigen exprimiert.

9. Genetisch verändertes abgeschwächtes Schweinegrippe-Virus nach einem der Ansprüche 1 bis 5, wobei das ab- 10 geschwächte Virus verändert ist, um ein Epitop von einem anderen Virus oder mindestens ein von einem anderen Virus abgeleitetes Segment zu exprimieren.

10. Immunogene Formulierung, umfassend das genetisch veränderte abgeschwächte Schweinegrippe-Virus nach einem der Ansprüche 1 bis 9, und einen physiologisch annehmbaren Arzneiträger. 15 11. Immunogene Formulierung nach Anspruch 10, wobei die Konzentration des abgeschwächten Virus in der immunogenen Formulierung ungefähr 104 bis ungefähr 107 pfu/ml ist.

12. Pharmazeutische Zusammensetzung, umfassend das genetisch veränderte abgeschwächte Schweinegrippe-Virus 20 nach einem der Ansprüche 1 bis 9 und einen physiologisch annehmbaren Arzneiträger.

13. Pharmazeutische Zusammensetzung nach Anspruch 12, wobei die Konzentration des abgeschwächten Virus in der pharmazeutischen Zusammensetzung ungefähr 104 bis ungefähr 1012 pfu/ml ist.

25 14. Genetisch verändertes abgeschwächtes Schweinegrippe-Virus, wie definiert in einem der Ansprüche 1 bis 9, zur Verwendung bei der Immunisierung oder dem Hervorrufen einer Immunantwort bei einem Schwein.

15. Genetisch verändertes abgeschwächtes Schweinegrippe-Virus wie definiert in einem der Ansprüche 1 bis 9 zur Verwendung bei der Prävention einer Erkrankung oder eines Symptoms verbunden mit einer Schweinegrippe- 30 Virusinfektion bei einem Schwein.

16. Genetisch verändertes abgeschwächtes Schweinegrippe-Virus wie definiert in einem der Ansprüche 1 bis 9 zur Verwendung bei der Behandlung einer Erkrankung verbunden mit einer Schweinegrippe-Virusinfektion bei einem Schwein. 35 17. Immunogene Formulierung wie definiert in Anspruch 10 oder 11 zur Verwendung bei der Immunisierung oder dem Hervorrufen einer Immunantwort bei einem Schwein.

18. Pharmazeutische Zusammensetzung wie definiert in Anspruch 12 oder 13 zur Verwendung bei der Prävention einer 40 Erkrankung oder eines Symptoms verbunden mit einer Schweinegrippe-Virusinfektion bei einem Schwein.

19. Pharmazeutische Zusammensetzung wie definiert in Anspruch 12 oder 13 zur Verwendung bei der Behandlung einer Erkrankung verbunden mit einer Schweinegrippe-Virusinfektion bei einem Schwein.

45 20. Kultivierte Zelle, enthaltend das genetisch veränderte abgeschwächte Schweinegrippe-Virus nach einem der An- sprüche 1 bis 9.

21. Kultivierte Zelle nach Anspruch 20, wobei die Zelle eine Schweinezelle oder eine Schweinezelllinie ist.

50 22. Kultivierte Zelle nach Anspruch 21, wobei die Schweinezelllinie PK(D1) Zellen, PK(15) Zellen, PK13 Zellen, SJPL Zellen, NSK Zellen, LLC-PK1 Zellen, LLC-PK1A Zellen, ESK-4 Zellen, ST Zellen, PT-K75 Zellen oder PK-2a/CL 13 Zellen ist.

23. Embryoniertes Ei, enthaltend das genetisch veränderte abgeschwächte Schweinegrippe-Virus nach einem der An- 55 sprüche 1 bis 9.

24. Embryoniertes Ei nach Anspruch 23, das von einem Huhn ist.

58 EP 2 497 492 B1

25. Embryoniertes Ei nach Anspruch 23 oder 24, wobei das embryonierte Ei 6, 7 oder 8 Tage alt ist.

26. Kultivierte Zelle nach einem der Ansprüche 20 bis 22 oder das embryonierte Ei nach einem der Ansprüche 23 bis 25, wobei die kultivierte Zelle oder das embryonierte Ei Interferon defizient ist. 5 27. Verfahren zur Herstellung einer immunogenen Formulierung oder einer pharmazeutischen Formulierung, umfassend:

(a) Vermehren des genetisch veränderten abgeschwächten Schweinegrippe-Virus nach einem der Ansprüche 10 1 bis 9 in

(i) der Zelle nach einem der Ansprüche 20 bis 22 oder 26, oder (ii) dem embryonierten Ei nach einem der Ansprüche 23 bis 26, und

15 (b) Sammeln der Virus-Nachkommen.

Revendications

20 1. Virus de la grippe porcine atténué génétiquement modifié, dans lequel le virus comprend un gène NS1 de virus de la grippe porcine avec une mutation ayant pour résultat une protéine NS1 de virus de la grippe porcine ayant une délétion de exactement 90 résidus d’acides aminés à partir de l’extrémité carboxy-terminale de NS1, dans lequel le gène NS1 du virus de la grippe porcine provient de A/Swine/Texas/4199-2/98.

25 2. Virus de la grippe porcine atténué génétiquement modifié selon la revendication 1, dans lequel le virus de la grippe porcine est H1N1, H1N2, ou H3N2.

3. Virus de la grippe porcine atténué génétiquement modifié selon la revendication 1, dans lequel le virus atténué est un virus réassorti. 30 4. Virus de la grippe porcine atténué génétiquement modifié selon la revendication 1, dans lequel le virus de la grippe porcine est A/Swine/Colorado/1/77, A/Swine/Colorado/23619/99, A/Swine/Cote d’Armor/3633/84, A/Swine/En- gland/195852/92, A/Swine/Finistere/2899/82, A/Swine/Hong Kong/10/98, A/Swine/Hong Kong/9/98, A/Swine/Hong Kong/81/78, A/Swine/Illinois/100084/01, A/Swine/Illinois/100085A/01, A/Swine/Illinois/21587/99, A/Swine/India- 35 na/1726/88, A/Swine/Indiana/9K035/99, A/Swine/Indiana/P12439/00, A/Swine/Iowa/30, A/Swine/Iowa/15/30, A/Swine/Iowa/533/99, A/Swine/Iowa/569/99, A/Swine/Iowa/3421/90, A/Swine/Iowa/8548-1/98, A/Swine/Io- wa/930/01, A/Swine/Iowa/17672/88, A/Swine/Italy/1513-1/98, A/Swine/Italy/1523/98, A/Swine/Korea/CY02/02, A/Swine/Minnesota/55551/00, A/Swine/Minnesota/593/99, A/Swine/Minnesota/9088-2/98, A/Swine/Nebras- ka/1/92, A/Swine/Nebraska/209/98, A/Swine/Netherlands/12/85, A/Swine/North Carolina/16497/99, A/Swine/North 40 Carolina/35922/98, A/Swine/North Carolina/93523/01, A/Swine/North Carolina/98225/01, A/Swine/Oedenro- de/7C/96, A/Swine/Ohio/891/01, A/Swine/Oklahoma/18717/99, A/Swine/Oklahoma/18089/99, A/Swine/Onta- rio/01911-1/99, A/Swine/Ontario/01911-2/99, A/Swine/Ontario/41848/97, A/Swine/Ontario/97, A/Swine/Que- bec/192/81, A/Swine/Quebec/192/91, A/Swine/Quebec/5393/91, A/Swine/Taiwan/7310/70, A/Swine/Ten- nessee/24/77, A/Swine/Texas/4199-2/98, A/Swine/Wisconsin/125/97, A/Swine/Wisconsin/136/97, A/Swine/Wis- 45 consin/163/97, A/Swine/Wisconsin/164/97, A/Swine/Wisconsin/166/97, A/Swine/Wisconsin/168/97, A/Swine/Wis- consin/235/97, A/Swine/Wisconsin/238/97, A/Swine/Wisconsin/457/98, A/Swine/Wisconsin/458/98, A/Swine/Wis- consin/464/98 ou A/Swine/Wisconsin/14094/99.

5. Virus de la grippe porcine atténué génétiquement modifié selon la revendication 4, dans lequel le virus de la grippe 50 porcine est A/Swine/Texas/4199-2/98.

6. Virus de la grippe porcine atténué génétiquement modifié selon l’une quelconque des revendications 1 à 5, dans lequel le virus atténué est un virus chimérique qui exprime une séquence hétérologue.

55 7. Virus de la grippe porcine atténué génétiquement modifié selon l’une quelconque des revendications 1 à 5, dans lequel le virus atténué est un virus chimérique qui exprime un épitope d’un pathogène étranger.

8. Virus de la grippe porcine atténué génétiquement modifié selon l’une quelconque des revendications 1 à 5, dans

59 EP 2 497 492 B1

lequel le virus atténué est un virus chimérique qui exprime un antigène tumoral.

9. Virus de la grippe porcine atténué génétiquement modifié selon l’une quelconque des revendications 1 à 5, dans lequel le virus atténué est modifié pour exprimer un épitope provenant d’un virus différent ou au moins un segment 5 dérivé d’un virus différent.

10. Formulation immunogène comprenant le virus de la grippe porcine atténué génétiquement modifié selon l’une quelconque des revendications 1 à 9, et un excipient physiologiquement acceptable.

10 11. Formulation immunogène selon la revendication 10, dans laquelle la concentration du virus atténué dans ladite formulation immunogène est d’environ 104 à environ 107 pfu/ml.

12. Composition pharmaceutique comprenant le virus de la grippe porcine atténué génétiquement modifié selon l’une quelconque des revendications 1 à 9, et un excipient physiologiquement acceptable. 15 13. Composition pharmaceutique selon la revendication 12, dans laquelle la concentration du virus atténué dans ladite composition pharmaceutique est d’environ 104 à environ 1012 pfu/ml.

14. Virus de la grippe porcine atténué génétiquement modifié tel que défini dans l’une quelconque des revendications 20 1 à 9, pour utilisation d’immunisation ou d’induction d’une réponse immunitaire chez un porc.

15. Virus de la grippe porcine atténué génétiquement modifié tel que défini dans l’une quelconque des revendications 1 à 9, pour utilisation dans la prévention d’une maladie ou d’un symptôme associé à une infection par le virus de la grippe porcine chez un porc. 25 16. Virus de la grippe porcine atténué génétiquement modifié tel que défini dans l’une quelconque des revendications 1 à 9, pour utilisation dans le traitement d’une maladie associée à une infection par le virus de la grippe porcine chez un porc.

30 17. Formulation immunogène telle que définie dans la revendication 10 ou 11, pour utilisation dans l’immunisation immuniser ou induire une réponse immunitaire chez un porc.

18. Composition pharmaceutique telle que définie dans la revendication 12 ou 13, pour utilisation dans la prévention d’une maladie ou d’un symptôme associé à une infection par le virus de la grippe porcine chez un porc. 35 19. Composition pharmaceutique telle que définie dans la revendication 12 ou 13, pour utilisation dans le traitement d’une maladie associée à une infection par le virus de la grippe porcine chez un porc.

20. Cellule cultivée contenant le virus de la grippe porcine atténué génétiquement modifié selon l’une quelconque des 40 revendications 1 à 9.

21. Cellule cultivée selon la revendication 20, dans laquelle la cellule est une cellule porcine ou une lignée de cellules porcines.

45 22. Cellule cultivée selon la revendication 21, la lignée de cellules porcines étant des cellules PK (D1), des cellules PK (15), des cellules PK13, des cellules SJPL, des cellules NSK, des cellules LLC-PK1, des cellules LLC-PK1A, des cellules ESK-4, des cellules ST, des cellules PT-K75 ou des cellules PK-2a/CL 13.

23. OEuf embryonnaire contenant le virus de la grippe porcine atténué génétiquement modifié selon l’une quelconque 50 des revendications 1 à 9.

24. OEuf embryonnaire selon la revendication 23 qui provient d’un poulet.

25. OEuf embryonnaire selon la revendication 23 ou 24, dans lequel l’oeuf embryonnaire a 6, 7, ou 8 jours. 55 26. Cellule cultivée selon l’une quelconque des revendications 20 à 22, ou oeuf embryonnaire selon l’une quelconque des revendications 23 à 25, dans lequel la cellule cultivée ou l’oeuf embryonnaire est déficient en interféron.

60 EP 2 497 492 B1

27. Procédé de production d’une formulation immunogène ou d’une formulation pharmaceutique comprenant:

(a) la propagation du virus de la grippe porcine atténué génétiquement modifié de l’une quelconque des revendications 1 à 9 dans 5 (i) la cellule de l’une quelconque des revendications 20 à 22 ou 26, ou (ii) l’oeuf embryonnaire de l’une quelconque des revendications 23 à 26, et

(b) la collecte de la descendance virale. 10

61 EP 2 497 492 B1

62 EP 2 497 492 B1

63 EP 2 497 492 B1

64 EP 2 497 492 B1

65 EP 2 497 492 B1

66 EP 2 497 492 B1

67 EP 2 497 492 B1

68 EP 2 497 492 B1

69 EP 2 497 492 B1

70 EP 2 497 492 B1

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 5166057 A [0101] • US 5820871 A [0142] • US 5854037 A [0101] • US 4444887 A [0144] • EP 0702085 A1 [0101] • US 4716111 A [0144] • US 152845 A [0101] • WO 9846645 A [0144] • WO 9712032 A [0101] • WO 9850433 A [0144] • WO 9634625 A [0101] • WO 9824893 A [0144] • EP 780475 A [0101] • WO 9816654 A [0144] • WO 9902657 A [0101] • WO 9634096 A [0144] • WO 9853078 A [0101] • WO 9633735 A [0144] • WO 9802530 A [0101] • WO 9110741 A [0144] • WO 9915672 A [0101] • US 6635246 B [0150] • WO 9813501 A [0101] • US 5891705 A [0150] • WO 9706270 A [0101] • US 5106619 A [0150] • EP O780475 A1 [0101] • US 4693981 A [0150] • WO 0104333 A [0102] • US 5962427 A [0167] • US 6649372 B [0102] • US 20020193338 A [0167] • US 6635416 B [0112] [0143] • WO 9308656 A [0188] • US 829711 A [0117] • US 6162432 A [0188] • US 6573079 B [0127] • WO 60576418 A [0261] • US 4873191 A, Hoppe & Wagner [0131]

Non-patent literature cited in the description

•R.A.LAMB; P.W. CHOPPIN. Annual Review of Bi- • NEMEROFF et al. Mol. Cell, 1998, vol. 1, 991-1000 ochemistry, 1983, vol. 52, 467-506 [0004] [0006] • GARCÍA-SASTRE. Microbes Infect, 2002, vol. 4, •QIU; KRUG. J. Virol., 1994, vol. 68, 2425-2432 647-55 [0005] [0006] • GARCÍA-SASTRE et al. Virology, 1998, vol. 252, •LUal.et Genes Dev., 1994, vol. 8, 1817-1828 [0006] 324-30 [0005] • GARCIA-SASTRE et al. Virology, 1998, vol. 252, •KINGal.et Virology, 1975, vol. 64, 378 [0006] 324-330 [0007] •QIUal.et RNA, 1995, vol. 1, 304 [0006] • EGOROV et al. J. Virol., 1998, vol. 72, 6437-6441 •QIUal.et J. Virol., 1994, vol. 68, 2425 [0006] [0007] • HATADA FUKUDA. J Gen Virol., 1992, vol. 73, •TALONal.et Proc. Natl. Acad. Sci. USA, 2000, vol. 3325-9 [0006] 97, 4309-4314 [0007] • FORTES et al. EMBO J., 1994, vol. 13, 704 [0006] • EASTERDAY. Philos Trans R Soc Lond B Biol Sci, • ENAMI et al. J. Virol., 1994, vol. 68, 1432 [0006] 1980, vol. 288, 433-7 [0008] • DE LA LUNA et al. J. Virol., 1995, vol. 69, 2427 • KIDA et al. J Gen Virol, 1994, vol. 75, 2183-8 [0008] [0006] • SCHOLTISSEK. Eur J Epidemiol, 1994, vol. 10, • LU et al. Genes Dev., 1994, vol. 8, 1817 [0006] 455-8 [0008] • PARK et al. J. Biol Chem., 1995, vol. 270, 28433 • OLSEN. Virus Res, 2002, vol. 85, 199-210 [0008] [0006] • KARASIN et al. J Clin Microbiol, 2002, vol. 40, • NEMEROFF et al. Mol.Cell, 1998,vol. 1, 1991 [0006] 1073-9 [0008] • CHEN et al. EMBO J., 1994, vol. 18, 2273-83 [0006] • KARASIN et al. Virus Res, 2000, vol. 68, 71-85 •QIANal.et RNA, 1995, vol. 1, 948-956 [0006] [0008] • DONELAN et al. J. Virol., 2003, vol. 77, 13257-66 • OLSEN et al. Arch Virol, 2000, vol. 145, 1399-419 [0006] [0008] • ARAGÓN et al. Mol. Cell Biol, 2000, vol. 20, • WEBBY et al. J Virol, 2000, vol. 74, 8243-51 [0008] 6259-6268 [0006]

71 EP 2 497 492 B1

• WEBBY et al. Philos Trans R Soc Lond B Biol Sci, • MERAZ et al. Cell, 1996, vol. 84, 431-442 [0133] 2001, vol. 356, 1817-28 [0008] • DURBIN et al. Cell, 1996, vol. 84, 443-450 [0133] • ZHOU. Vet Microbiol, 2000, vol. 74, 47-58 [0008] [0138] • OLSEN et al. Arch Virol., 2000, vol. 145, 1399-419 • ADRA et al. Gene, 1987, vol. 60, 65-74 [0134] [0008] • SORIANO et al. Cell, 1991, vol. 64, 693-701 [0134] • GARCIA-SASTRE. Microbes Infect, 2002, vol. 4, •THOMAS; CAPECCHI. Cell, 1987, vol. 51, 503-512 647-55 [0009] [0134] • GARCIA-SASTRE et al. Virology, 1998, vol. 252, • GOSSLER et al. Proc. Natl. Acad. Sci. USA, 1986, 324-30 [0009] vol. 83, 9065-9069 [0137] • SHOPE, R.E. J. Exp. Med., 1931, vol. 54, 373-385 • MERAZ et al. Cell, 1996, vol. 84, 431 [0138] [0010] • JERNE. Ann. Immunol. (Paris), 1974, vol. 125c, 373 • OLSEN et al. Arch. Virol., 2000, vol. 134, 1399-1419 [0146] [0010] • JERNE et al. EMBO J., 1982, vol. 1, 234 [0146] • KARASIN et al. J. Clin. Microbiol., 2000, vol. 38, • KRUG et al. Textbook of Influenza. 1998, 82-92 2453-2456 [0010] [0153] • KARASIN et al. J. Clin Microbiol., 2002, vol. 40, • SEFTON. CRC Crit. Ref. Biomed. Eng., 1987, vol. 1073-1079 [0010] 14, 201 [0168] • KARASIN et al. J. Virol., 2000, vol. 74, 9322-9327 • BUCHWALD et al. Surgery, 1980, vol. 88, 507 [0168] [0010] • SAUDEK et al. N. Engl. J. Med., 1989, vol. 321, 574 • Orthomyxoviruses. MURPHY, B.R. ; R.G. WEB- [0168] STER. Fields Virology. Lippincott-Raven, 1996, • Medical Applications of Controlled Release. CRC 1397-1445 [0013] Pres, 1974 [0168] • SHU et al. Virology, 1994, vol. 202, 825-33 [0016] • Controlled Drug Bioavailability, Drug Product Design • SCHOLTISSEK. Med. Principles Pract., 1990, vol. 2, and Performance. Wiley, 1984 [0168] 65-71 [0016] • RANGER ; PEPPAS. J. Macromol. Sci. Rev. Macro- • ZHOU et al. J Virol., 1999, vol. 73, 8851-6 [0016] mol. Chem., 1983, vol. 23, 61 [0168] • GORSE ; BELSHE. J. Clin. Microbiol., 1990, vol. 28, • LEVY et al. Science, 1985, vol. 228, 190 [0168] 2539-2550 [0017] • DURING et al. Ann. Neurol., 1989, vol. 25, 351 [0168] • GORSE et al. J. Infect. Dis., 1995, vol. 172, 1-10 • HOWARD et al. J. Neurosurg., 1989, vol. 71, 105 [0017] [0168] • KRUG ; NICHOLSON et al. Textbook of Influenza. • GOODSON. Medical Applications of Controlled Re- 1998, 82-92 [0100] lease, 1984, vol. 2, 115-138 [0168] • FODOR et al. J. Virol., 1999, vol. 73, 9679-9682 •LANGER. Science,1990, vol. 249, 1527-1533 [0168] [0102] •TAYLOR. Pig Diseases. Diamond Farm Book Pubns, • HOFFMANN et al. Proc. Natl. Acad. Sci. USA, 2000, 1995 [0173] [0199] vol. 97, 6108-6113 [0102] [0225] [0229] [0230] • STRAW et al. Diseases of Swine. Iowa State Univer- • NEUMANN et al. Proc. Natl. Acad. Sci. USA, 1999, sity Press, 1999 [0173] [0199] vol. 96, 9345-9350 [0102] • Physicians’ Desk Reference. 2004 [0177] [0182] • BLUYSSEN et al. Eur. J. Biochem., 1994, vol. 220, [0185] 395-402 [0116] [0207] • Merck Veterinary Manual. 1998 [0177] [0185] [0195] • KRISHNAN et al. Eur. J. Biochem., 1997, vol. 247, [0199] 298-305 [0116] [0140] [0206] • Merck.Veterinary Manual. 1998 [0182] • SANG. Trends Biotechnol., 1994, vol. 12, 415 [0131] • Cecil Textbookof Medicine. 1988 [0182] • PERRY et al. Transgenic Res., 1993, vol. 2, 125 • Physician’s Desk Reference. 2004 [0195] [0131] • STRAW et al. Diseases of the Swine. Iowa State Uni- • STERN, C.D. Curr Top Microbiol Immunol, 1996, vol. versity Press, 1999 [0195] 212, 195-206 [0131] • Goodman & Gilman’s The Pharmacological Basis Of • SHUMAN. Experientia, 1991, vol. 47, 897 [0131] Basis Of Therapeutics. Mc-Graw-Hill, 2001 [0199] • VAN DER PUTTEN et al. Proc. Natl. Acad. Sci., USA, • Physicians’ Desk Reference (PDR). Medical Eco- 1985, vol. 82, 6148-6152 [0131] nomics Co., Inc, 2004 [0199] • THOMPSON et al. Cell, 1989, vol. 56, 313 [0131] • PALMER et al. Advanced laboratory techniques for •LO. Mol Cell. Biol., 1983, vol. 3, 1803 [0131] influenza diagnosis. U.S. Department of Health, Ed- • LAVITRANO et al. Cell, 1989, vol. 57, 717 [0131] ucation and Welfare Immunology Series. U.S. De- • GORDON. Transgenic Animals, Intl. Rev. Cytol., partment of Health, Education and Welfare, 1975 1989, vol. 115, 171 [0131] [0218] • CAPECCHI. Science, 1989, vol. 244, 1288 [0132] •STOJDL. Cancer Cell, 2003, vol. 4, 263-75 [0224] • MANSOUR et al. Nature, 1988, vol. 336, 348-352 •FODORal.et J Virol, 1999, vol. 73, 9679-82 [0229] [0132]

72 EP 2 497 492 B1

• NEUMANN et al. Proc NatI Acad Sci USA, 1999, vol. • PALMER. Education, and Welfare. U.S. Department 96, 9345-50 [0229] of Health, 1975, 51-52 [0242] • PARK et al. J. Virol., 2003, vol. 70, 9522-9532 [0236] •MENGELING. Am J Vet Res, 1996, vol. 57, 834-839 • DONELAN. J Virol, 2003, vol. 77, 13257-66 [0236] [0242] [0238] • REED. Am J Hyg, 1938, vol. 27, 493-497 [0244]